Cosmetic methods and products

ABSTRACT

This invention relates to method of improving the appearance of skin or hair. In particular, it relates to cosmetic products such as creams and serums comprising a subject&#39;s own platelet rich plasma (PRP). The invention also relates to kits that allow the user to apply the cosmetic products daily for several days.

CROSS REFERENCE TO RELATED APPLICATION

The present application is a divisional of U.S. patent application Ser.No. 15/323,702, filed Jan. 3, 2017, which is a national phase entryunder 35 U.S.C. § 371 of International Application No.PCT/GB2015/051746, filed Jun. 12, 2015, entitled “COSMETIC METHODS ANDPRODUCTS,” which designated, among the various States, the United Statesof America, and which is hereby incorporated by reference.

This invention relates to methods of improving the appearance of skin orhair. In particular, it relates to cosmetic products such as creams andserums comprising a subject's own platelet rich plasma (PRP), a fractionof plasma which is particularly rich in platelets, and uses thereof. Italso relates to uses of cosmetic products, such as creams and serumscomprising a subject's own platelet rich plasma in conjunction withtreatment to improve the appearance of the skin or hair.

Ageing causes tissue repair and cell regeneration to slow down. Skinageing is caused by a slowing down of the skin renewal cycles over theyears exacerbated by damage to the skin due to stress, photo-exposure,pollution, exposure to cigarette smoke and unhealthy lifestyle factors.The skin aging process is marked by very distinct signs: the appearanceof wrinkles, a variation in the skin pigmentation, loss of elasticityand compactness, and the relaxing of the tissues. Skin aging influencesthe three main skin structures: epidermis, dermal-epidermal junction anddermis. In particular, among the various factors which significantlyparticipate in the skin aging process, is a reduction in the thicknessof the dermis. The dermis is a connective tissue having a thickness of3-4 mm, beneath the epidermis, consisting of a cell population dispersedin an abundant intercellular matrix. The majority of dermal cells arefibroblasts, destined for the synthesis of collagen and elastic fibres.The former have the function of support and resistance, the elasticfibres ensure a correct elasticity of the skin whereas the intercellularmatrix in which they are immersed has a strengthening function. As weage the renewal of these important structures slows down and this leadsto the signs such as uneven skin tone and wrinkles that many peoplewould like to avoid in order to have a more youthful appearance.

Plasma comprises a multitude of molecules and cells that are responsiblefor regulating key process involved in tissue repair, includingproliferation, chemotaxis. Platelets, initially known to be responsiblefor blood clotting, release growth factors including Platelet-derivedgrowth factor (PDGF), a potent chemotactic agent, and TGF beta, whichstimulates the deposition of extracellular matrix. Both of these growthfactors have been shown to play a significant role in the repair andregeneration of connective tissues. The concentrated platelets in PRPdeliver an increased number of growth factors to the topical area andactivation of the platelets, for example with calcium chloride releasesthe majority of growth factors from the platelets.

The seven principal known growth factors in platelets are: PlateletDelivered Growth Factors such as (PDGFaa), (PDGFbb), (PDGFab),Transforming Growth Factor beta-, (TGF-b), TGF-b2, Vascular EndothelialGrowth Factor (VEGF) and Epithelial Growth Factor

As a concentrated source of platelets, plasma contains several cytokinesthat stimulate tissue regeneration and have an anti-ageing activity.Plasma has received popular attention due to its use in treating sportsinjuries in professional athletes and it is also known for use indentistry and wound healing. Plasma therapy, involves centrifuging aperson's blood until the blood gets separated in its main compounds: redblood cells, white blood cells and plasma. The richer part of plasma iscalled PRP or F2 fraction (F2) and the poorer part of plasma is calledPPP or F1 fraction (F1). Growth factors can be released by activatingthe plasma and then injected into the injured tissue.

Plasma, PPP and PRP have been used in a range of applications byinjecting it into the skin to reduce the visible signs of ageing andalso in medical treatments for wound healing and speeding the repair ofdamaged tissues. Most medical and cosmetic uses of plasma are done byactivating the plasma using calcium chloride to release the growthfactors from the platelets and injecting activated plasma into the skinor damaged tissue.

Activated plasma has been injected into the dermal layer of the skin inorder to reduce the signs of ageing but this treatment can be painfuland lead to redness, soreness and bruising of the skin. The effects ofthe plasma only last for a few weeks or months and the treatment must berepeated in order to prolong the effect. Various other anti-ageingtreatments, such as cosmetic laser treatments are known to have positiveeffects on the appearance of the skin but also can cause skinirritation, pain and redness in the short term.

It would therefore be advantageous to provide a topical formulation ofPRP that could be used to improve the appearance of skin and also can beused after other cosmetic treatments, such as injected PRP and cosmeticlaser treatment, to prolong and enhance the effects of the cosmetictreatments so that they could be carried out less frequently.

The object of the present invention is therefore to provide a skin-careproduct that makes it possible to apply PRP to the skin topically eitheras a cosmetic treatment alone or after a first cosmetic treatment suchas injected PRP treatment or cosmetic laser treatment to prolong andenhance the cosmetic effects of the first cosmetic treatment.

In a first aspect the present invention provides a method of improvingthe cosmetic appearance of skin and/or hair of a subject wherein themethod comprises,

-   (a) Providing a sample of PRP from the subject;-   (b) Providing a cosmetically acceptable carrier;-   (c) Admixing a portion of the PRP to a portion of the cosmetically    acceptable carrier to form a topical composition;-   (d) Applying the topical composition to an area of the subject's    skin.-   (e) Repeating steps (c) and (d)

Steps c and d may be repeated every day for up to 8 days. After 8 daysfresh PRP may be provided from the subject to continue the treatment. Anadvantage of the present method is that a portion of PRP may be mixedwith activator each day immediately before it is applied to the skin.This means that growth factors are kept inside the platelets, whichprotects them from degradation, until the activator is mixed with PRP.The growth factors are then released from the platelets into thecomposition. The advantage of this method is that it allows thetreatment to be used every day with fresh growth factors that have justbeen released from the platelets immediately before application to theskin. These advantages are provided without having to provide a newsample of PRP from the subject each day. Therefore, the subject cancontinue the treatment at home for 8 days using freshly activated PRPevery day.

Various authors have discussed the use of a patient's own plasma inwound healing and in cosmetic procedures and there is varied andinconsistent use of a number of terms in this field. In this applicationthe terms PRP and F2 are used to describe the richer fraction of plasmathat contains a higher number of platelets and the terms PPP and F1 areused to describe the poorer fraction of the plasma that contains a lowernumber of platelets. This is shown in FIGS. 27-29. The methods describedherein may, preferably be performed using the PRP fraction of plasma.This is advantageous because the higher number of platelets provides aricher source of growth factors and skin enhancing factors.

In contrast to the use in this application, other authors have used theterm PRP to describe the whole plasma fraction of the blood rather thanjust the fraction of plasma that is rich in platelets.

An activator may be admixed with the PRP within less than 4 hours,within less than 3 hours, within less than 2 hours, within less than 1hour, within less than 30 minutes, within less than 20 minutes, withinless than 10 minutes or within less than 8 minutes before the topicalcomposition is applied to the subject's skin.

Preferably an activator may be admixed with PRP immediately before thetopical composition is applied to the skin. Immediately before means afew minutes or seconds before, for example, immediately before may meanthe PRP is admixed with an activator less than 5 minutes, less than 2minutes, less than one minute or less than 30 seconds before applyingthe mixture to the skin. This is advantageous because platelets in thePRP may be activated and release growth factors immediately beforeapplying the composition to the skin so that the growth factors do notdegrade before they are applied to the skin and there may be a highlevel of growth factors in the composition.

The cosmetically acceptable carrier may comprise an activator and thetopical composition may be applied to the area of the subject's skinimmediately after admixing the cosmetically acceptable carriercomprising activator with the PRP.

The topical composition may be applied to the area of the subject's skinwithin less than 4 hours, within less than 3 hours, within less than 2hours, within less than 1 hour, within less than 30 minutes, within lessthan 20 minutes, within less than 10 minutes or within less than 8minutes of the activator mixing with the PRP.

Preferably the cosmetically acceptable carrier may be admixed with PRPimmediately before the topical composition is applied to the skin.Immediately before means a few minutes or seconds before, for example,immediately before may mean the PRP is admixed with the cosmeticallyacceptable carrier less than 5 minutes, less than 2 minutes, less thanone minute or less than 30 seconds before applying the mixture to theskin. This is advantageous because platelets in the PRP may be activatedand release growth factors immediately before applying the compositionto the skin so that the growth factors do not degrade before they areapplied to the skin and there may be a high level of growth factors inthe composition.

The PRP may be stored separately from the cosmetically acceptablecarrier comprising an activator and the PRP may be mixed with thecosmetically acceptable carrier comprising an activator immediatelybefore applying the composition to the skin.

In some embodiments the cosmetically acceptable carrier may not comprisean activator and an activator may be admixed with the PRP and thecosmetically acceptable carrier immediately before application of themixture to the skin.

If the activator is not in the cosmetically acceptable carrier the PRPmay be mixed with the cosmetically acceptable carrier soon after it ismade and stored as a composition comprising PRP and the cosmeticallyacceptable carrier. This is advantageous because the cosmeticallyacceptable carrier may comprise ingredients that stabilise or preservethe PRP and/or platelets in the PRP. An activator may be storedseparately from the composition comprising the cosmetically acceptablecarrier and PRP. The activator may be admixed with the compositionimmediately before applying the composition to the skin.

Step (e) or steps (c) and (d) may be repeated at least once a day, atleast twice a day, at least three times a day, at least four times a dayor at least six times a day.

Step (e) or steps (c) and (d) may be repeated for at least two days, atleast three days, at least four days, at least five days, at least sixdays, at least seven days, at least eight days, at least nine days or atleast ten days.

Step (e) may be repeated at least once a day for at least eight days.

The activator may be collagen. This is advantageous because collagen isnaturally occurring in the skin and therefore is not likely to causeadverse reactions in the skin. The activator may not be calciumchloride. This is advantageous because calcium chloride can causeadverse reactions and/or be irritating to the skin.

The method may be performed on an area of the subject's skin that hasbeen treated using PRP injection or another cosmetic treatment such ascosmetic laser skin treatments.

The method may comprise, before or after step (c), a further step (f)injecting a portion of the platelet rich plasma into one or more areasof the subject's skin. When the method is used in conjunction withinjecting PRP into an area of the subject's skin the topical compositionis applied to at least the same area of the subject's skin. When themethod is used in conjunction with injecting PRP into an area of thesubject's skin the topical composition may be applied to at least thesame area of the subject's skin immediately after or within an hourafter the injections have been performed and then the topicalcomposition may be applied to the same area of skin repeatedlythereafter.

According to a further aspect of the invention we provide a method forimproving the appearance of the skin and/or hair of a subject comprisingthe steps of:

-   -   (g) Providing a sample of platelet rich plasma from the subject;    -   (h) Injecting a first portion of platelet rich plasma into one        or more areas of the subject's skin;    -   (i) Combining a second portion of platelet rich plasma with a        cosmetically acceptable carrier to provide a topical        composition;    -   (j) Applying the topical composition from step (i) to the one or        more areas of the subject's skin where the platelet rich plasma        was injected in step (h).

Platelet rich plasma may be provided by centrifuging a sample of bloodfrom a person in order to separate the platelets from the other bloodcells and taking a fraction comprising plasma with a high concentrationof platelets.

The PRP may comprise more than 10,000 platelets/μl, more than 10,000platelets/μl, more than 10,000 platelets/μl, more than 10,000platelets/μl, more than 10,000 platelets/μl, more than 100,000platelets/μl, more than 500,000 platelets/μ1 or more than 900,000platelets/μl. The PRP may comprise Platelet Delivered Growth Factorssuch as (PDGFaa), (PDGFbb), (PDGFab), Transforming Growth Factor beta-,(TGF-b), TGF-b2, Vascular Endothelial Growth Factor (VEGF) andEpithelial Growth Factor (EGF).

The PRP may be autologous, i.e. taken from a subject and injected backinto the same subject. This is advantageous because it avoids any riskof disease transmission between a donor and recipient and also avoidsthe possibility of reactions to foreign tissues or different ABO bloodantigens. It also avoids allergic or adverse reactions.

The area of skin where the topical composition is applied may be on anypart of the body. In particular the area of skin where the topicalcomposition is applied may be on the face, scalp, neck, chest, hands,arms, legs, abdomen or buttocks. It is advantageous to apply the topicalcomposition to areas where ageing skin causes concern such as the faceand neck. It is also advantageous to apply the topical composition tothe scalp because it can prolong the life of hair follicles and increasehair growth. For use on the scalp the topical composition may be hairGel, hair lotion or leave in conditioner. The topical composition may beformulated for use after PRP injection into the scalp. The topicalcomposition may be formulated for use with other hair-restoringtreatments.

The portion of the platelet rich plasma that is injected into the skinmay be activated by mixing it with activating agents such as calciumchloride or collagen before injection into the skin. Adding calciumchloride or collagen to the PRP causes the platelets to release most ofthe growth factors very quickly. This is advantageous because itprovides a high concentration of growth factors in the area where thePRP is injected.

The portion of PRP that is reserved for admixing with the cosmeticallyacceptable carrier may have an activator such as calcium chloride orcollagen added to it.

The activator may be collagen. This is advantageous because collagen isnaturally occurring in the skin and therefore is not likely to causeadverse reactions in the skin. The activator may not be calciumchloride. This is advantageous because calcium chloride can causeadverse reactions and/or be irritating to the skin.

The portion of PRP that is reserved for admixing with the cosmeticallyacceptable carrier may have no activator added to it. This isadvantageous because calcium chloride can be irritating to the skin.This is further advantageous because the growth factors are preservedinside the platelets until the platelets are activated.

Portions of PRP without activator may be added to a cosmeticallyacceptable carrier that comprises an activator shortly before beingapplied to the skin, for example before being applied to the skin. It isadvantageous to addmix a portion of PRP without activator and a portionof a cosmetically acceptable carrier that comprises activatorimmediately before applying the mixture to the skin. For example theymay be mixed a few seconds to or up to two minutes before applying tothe skin. This ensures that the growth factors are released from theplatelets just before the cosmetic composition is applied to the skin sothat the growth factors are as active as possible when they are appliedto the skin. The PRP may be stored without an activator for severaldays, for example at least 2 days, at least three days, at least fourdays, at least five days, at least six days, at least seven days, atleast eight days, at least nine days at least ten days or at least twoweeks and then added to the cosmetically acceptable carrier comprisingan activator which makes platelets release growth factors quickly aftermixing. PRP may be stored at room temperature, for example at about 22°C. PRP may not be stored in a refrigerator (for example at about 4° C.because cold storage will activate platelets during storage rather thanwhen mixed with an activator. PRP may also be stored frozen but theplatelets are damaged by freezing and release the growth factors shortlyafter the PRP is thawed. If freezing is used the PRP may be frozen inportions and each portion may be thawed immediately before applicationto the skin to avoid degradation of the growth factors. PRP may bestored for several weeks or several months frozen. When frozen PRP isthawed it may be mixed with the cosmetically acceptable carrier andapplied to skin immediately because growth factors may be released fromthe platelets as the thawing process may act as an activator.

The cosmetically acceptable carrier may comprise an activator. Thecosmetically acceptable carrier may comprise calcium chloride. Thecosmetically acceptable carrier may comprise collagen. The cosmeticallyacceptable carrier may comprise an activator such as collagen, solublecollagen, or hydrolyzed collagen that is less likely than calciumchloride to irritate the skin or cause adverse reactions. Collagen ispresent in the skin and therefore provides a more natural activator thancalcium chloride. Collagen may act as a gentler activator causing growthfactors to be released from the platelets at a slower rate than calciumchloride does. It is therefore suitable for use in the cosmeticallyacceptable carrier of the present invention. The topical composition maycomprise an activator that is not calcium chloride.

The activator may be collagen, soluble collagen or hydrolysed collagenand may be from any source such as animal collagen, bovine collagen orsynthetic collagen.

Advantageously, the cosmetically acceptable carrier may comprise anactivator and be stored separately from the PRP. A portion ofcosmetically acceptable carrier may be mixed with a portion of PRPwithin 1 hour, within 30 minutes, within 20 minutes or within 10 minutesbefore the composition is applied to the skin. Preferably a portion ofcosmetically acceptable carrier may be mixed with a portion of PRP andapplied to the skin immediately or within a few seconds up to 2 minutesof mixing. Where the cosmetically acceptable carrier comprises anactivator the activator may be calcium chloride or collagen. Collagenmay be an advantageous activator for inclusion in a cosmeticallyacceptable carrier because it may not cause irritation of the skin.

Each portion of PRP and cosmetically acceptable carrier may be justenough for one application. This is advantageous because just enough PRPand cosmetically acceptable carrier may be mixed for one application andthe mixture applied to the skin immediately, for example within a fewseconds or up to two minutes. This means that the growth factors arereleased from PRP immediately before application to the skin so thatthey are fresh and active when they are applied to the skin. Theportions of PRP and cosmetically acceptable carrier may be roughly equalin volume so that it is simple to make a 1:1 mixture of the twocomponents.

The cosmetically acceptable carrier may comprise or consist of a cream,gel, serum, balm, sun cream, after sun cream, foundation, tinted cream,tinted sun cream, soothing anti redness cream with green tint, scalpserum, solution, suspension, emulsion, ointment, foam, paste, lotion,powder, soap, surfactant-containing cleansing oil or spray.

The cosmetically acceptable carrier may advantageously be a cream, gel,lotion or a serum.

The cosmetically acceptable carrier may comprise at least one ingredientto increase penetration of the platelet rich plasma into the skin. Acomposition that comprises at least one ingredient that increasespenetration into the skin may be a serum. A serum may also have aconsistency, lipid content and/or hydrophobicity that increases theamount of penetration into the skin.

The cosmetically acceptable carrier may comprise or consist of adressing, adhesive bandage, gauze, or a facial mask. PRP may be soakedinto a facial mask that is applied to the skin after PRP has beeninjected. This can reduce irritation, swelling, pain and redness of theskin at the site where the PRP has been injected.

The cosmetically acceptable carrier may comprise the ingredients fromtable 4 and at least one preservative from table 5.

The carrier may comprise at least one humectant, at least one emollientand at least one emulsifier.

The cosmetically acceptable carrier may be suitable for preserving andstabilising platelet rich plasma that is not mixed with calcium chlorideor collagen.

The cosmetically acceptable carrier may not comprise an activator. Thecosmetically acceptable carrier may not comprise calcium chloride. Thecosmetically acceptable carrier may comprise an activator.

The cosmetically acceptable carrier may comprise a suitable amount ofcollagen to act as an activator of platelets.

The cosmetically acceptable carrier may not comprise calcium chloride.The cosmetically acceptable carrier may not comprise any activatorexcept for collagen.

The cosmetically acceptable carrier may comprise at least one ingredientto increase penetration of the platelet rich plasma or growth factorsinto the skin.

The cosmetically acceptable carrier may further comprise one or moreingredients from table 1 or table 2.

The cosmetically acceptable carrier may further comprise one or moreingredients selected from monocytes, stem cells, gene therapy products,vitamins, palmitate retinol, tocoferil acetate, sodium ascorbilphosphate, D-panthenol, peptides, recombinant growth factors, micronizedhuman-identical hormones, aminoacids, phyto-extracts, anti-oxidants,lipoic acid, DMAE, collagen, GAG, hyaluronic acid, proteoglycans,adenine, guanine, cytosine, thimine, trace elements, minerals,proteases, ceramides, polisaccarides, algae and marine extracts.

In another aspect the present invention provides a cosmetic compositioncomprising the cosmetically acceptable carrier and platelet rich plasma.Between 10 and 20 ml of PRP may be diluted with cosmetic composition upto a final volume of 50 ml.

The platelet-rich plasma concentration in the cosmetic composition maybetween 0.1 and 50% (w/w), between 1 and 50%, between 2 and 30%, between4 and 10% or between 30 and 50% of the total weight of the composition.

The PRP may have no activator added. The cosmetically acceptable carriermay comprise no activator. The cosmetic composition may comprise noactivator.

The cosmetic composition may comprise active growth factors for at least1 day, at least 2 days, at least 3 days, at least 4 days, at least 5days, at least 6 days, at least 7 days, at least 10 days, at least 2weeks or at least 3 weeks.

In the method of the present invention the step of applying the cosmeticcomposition to the area of skin where PRP has been injected may berepeated at least twice, at least three times, at least four times, atleast five times, at least six times, at least seven times, at leasteight times or at least nine times.

In the method of the present invention the step of applying the cosmeticcomposition to the area of skin where PRP has been injected may berepeated at least once every day, at least twice daily, at least threetimes daily, at least four times daily. The step of applying thecosmetic composition to the area of skin where the PRP has been injectedmay be repeated once or twice daily for up to 8 days.

In the method of the present invention the step of applying the cosmeticcomposition to the area of skin may be repeated for at least two days,at least three days, at least four days, at least six days, at leastseven days, at least 10 days, at least two weeks, at least three weeks,at least four weeks, at least six weeks or at least 8 weeks.

This is advantageous because, after the PRP injection treatment,applying the cosmetic composition comprising PRP may reduce immediatesoreness, redness, swelling or bruising. This is also advantageousbecause, after PRP injection treatment, applying the cosmeticcomposition comprising PRP may prolong and/or enhance the cosmeticeffect of the PRP injection treatment.

In a further aspect, the present invention provides a kit suitable foruse in performing the method of the present invention.

The kit may comprise one or more containers comprising chambers suitablefor storing PRP for one or more days. The one or more containers may besterile. The one or more chambers may be arranged to allow the PRP to beintroduced into the container under sterile conditions. The Kit maycomprise one or more portions of cosmetically acceptable carrier. Thekit may be arranged to allow mixing of a portion the PRP from the PRPcontainer with a portion of the cosmetically acceptable carrier. The kitmay be arranged so that a portion of PRP and a portion of cosmeticallyacceptable carrier can be mixed to provide a PRP composition and thecomposition can be applied to the skin.

This is advantageous because the cosmetically acceptable carrier maycomprise an activator. When a portion of PRP and a portion ofcosmetically acceptable carrier are mixed the platelets in that portionof PRP may be activated and release growth factors. The remainder of thePRP in the container may remain un-activated and may preserve the growthfactors it contains. Freshly activated PRP may therefore be appliedevery day for several days.

The kit may comprise a suitable container for storing PRP, one or moreportions of the cosmetically acceptable carrier comprising collagen asan activator and instructions for use. The user may then admix a portionof PRP with a portion of cosmetically acceptable carrier immediatelybefore applying the mixture to the skin. The container for storing PRPmay be designed to exclude airflow into the container. The container forstoring PRP may be sterile inside.

For example, the kit may comprise 8 ampules each containing a portion ofcosmetically acceptable carrier including collagen, a container for thePRP and instructions for use.

The kit may further comprise a mini vibration table/Agitator and theinstruction for use.

An agitator may be advantageous because it is advantageous to store thePRP with agitation to prolong the life of the platelets. Platelets thathave been stored with agitation have a greater amount of growth factorson day 8 than platelets that have not been stored with agitation. Thekit may also be adapted for storage at room temperature as storage atroom temperature improves the storage time of the platelets and meansthat there are more growth factors on day 8 of storage.

The kit may further comprise instructions for use. The instructions foruse may be use in a method of the present invention.

The container may be sterile. The container may be arranged to allowoxygen penetration into the container. The container may be arranged notto allow oxygen penetration into the container. The kit may comprise acontainer according to the present invention comprising a cosmeticallyacceptable carrier in at least one chamber and at least one chamberarranged to accept a portion of PRP. The kit may further comprisesuitable equipment for drawing blood, suitable containers for holdingblood while it is centrifuged, suitable equipment for removing a PRPfraction from the blood and/or suitable equipment for injecting PRP intothe skin.

In another aspect the present invention provides a container comprisinga first chamber and a second chamber arranged so that contents of thefirst chamber and contents of the second chamber are mixed as they exitthe container. The container may be sterile. A cosmetically acceptablecarrier contained in the first chamber and the second chamber isarranged to accept platelet rich plasma. The container may be sterile. Aportion of PRP may be put into the second chamber of the container. ThePRP may be stored in the second chamber of the container until the useris ready to apply the cosmetic composition. When the container is used aportion of the cosmetically acceptable carrier in the first chamber anda portion of the PRP in the second chamber leave the container and aremixed as they leave the container to provide a cosmetic compositioncomprising the cosmetically acceptable carrier and PRP. The cosmeticallyacceptable carrier and the PRP may be mixed in suitable proportions toprovide a cosmetic composition of the invention.

The cosmetically acceptable carrier may be contained in a first chamberof the container and PRP may be contained in a second chamber of thecontainer. This is advantageous because the carrier may comprise anactivator, for example calcium chloride, collagen or another activator.The PRP may remain in a chamber of the container and not be activatedfor several days so growth factors remain inside the platelets in thePRP. When the cosmetic composition is required the container may beactivated and carrier may be mixed with PRP. The platelets in the PRPmay release growth factors when they are mixed with the carrier thatcomprises an activator and therefore the resulting cosmetic compositionmay comprise a high level of active growth factors. This arrangement isadvantageous because the growth factors may remain active inside theplatelets for several days before being released when the PRP is mixedwith a carrier comprising an activator. The container may be arranged sothat a portion of the PRP and a portion of the carrier may be releasedat each use of the container. PRP may be prepared once at the time thatblood is taken from a subject and put into one chamber of the container.The PRP can thereby be stored without activation until needed and only aportion of the stored PRP may be mixed with carrier including activatoreach day before applying the composition to the skin.

The cosmetically acceptable carrier that does not comprise an activatormay be contained in a first chamber of the container and PRP may beadded to the cosmetically acceptable carrier. An activator may becontained in a second chamber of the container. This is advantageousbecause the carrier may comprise ingredients that stabilise PRP and/orstabilise growth factors inside the platelets in the PRP. An activator,for example calcium chloride, collagen or another activator may becontained in a second chamber of the container. The PRP and cosmeticallyacceptable carrier may remain in a chamber of the container and the PRPmay not be activated for several days so growth factors remain insidethe platelets in the PRP. When the cosmetic composition is required thecontainer may be activated and the activator may be mixed withcosmetically acceptable carrier and PRP. The platelets in the PRP mayrelease growth factors when they are mixed with the activator andtherefore the resulting cosmetic composition may comprise a high levelof active growth factors. This arrangement is advantageous because thegrowth factors may remain active inside the platelets for several daysbefore being released when the PRP is mixed with an activator. It isalso advantageous because the PRP is mixed with a cosmeticallyacceptable carrier soon after it is made and the cosmetically acceptablecarrier can stabilise and preserve the PRP until it is mixed with anactivator. The container may be arranged so that a portion of the PRPmixed with cosmetically acceptable carrier and a portion of theactivator may be released at each use of the container. PRP may beprepared once at the time that blood is taken from a subject and putinto one chamber of the container with the cosmetically acceptablecarrier. The PRP can thereby be stored without activation until neededand only a portion of the stored PRP may be mixed with activator eachday before applying the composition to the skin.

In order to prolong the life of the platelets and preserve the growthfactors inside them it is advantageous to store the PRP at roomtemperature. It is also advantageous to store the PRP so that it isagitated. It is further advantageous to store the PRP with low oxygenand minimal airflow. It is further advantageous to mix an activator withthe PRP immediately before applying the mixture to the skin so that thegrowth factors are released from the platelets immediately before themixture is applied to the skin. The kit is arranged to provide all ofthese features by providing a storage container for the PRP that isairtight and sterile, providing a device for agitating the PRP in thecontainer during storage. The container may be suitable for storage atroom temperature. The activator may be included in the cosmeticallyacceptable composition so that it can be mixed with the PRP immediatelybefore applying to the skin.

There now follows by way of example only a detailed description of thepresent invention with reference to the accompanying drawings, in which;

FIG. 1 shows a view of an embodiment of a container of the presentinvention.

FIG. 2 shows a view of an embodiment of a container of the presentinvention, the whole container is shown in FIG. 2a , embodiments of achamber suitable for containing PRP is shown in FIGS. 2b -d.

FIG. 3 shows an embodiment of a chamber suitable for containing PRP.

FIG. 4 shows pictures of the treated areas of patients 1, 2 and 3 fromgroup 1 and patients 1, 2 and 3 from group 2 showing a comparison of theskin before the treatment and after the treatment. Group 1 had PRPinjections in the treated area followed by using serum mixed with PRPeach day for 16 days. Group 2— only used serum mixed with PRP each dayfor 24 days on one side of their face (fresh blood was taken every 8days) Group 3-used the collagen serum only (without adding PRP to it).

FIG. 5 shows Light transmittance platelet aggregometry. The aggregometermeasures changes in light transmission of a stirred platelet suspensionexposed to a platelet agonist. It is calibrated by a cuvette containingplatelet poor plasma which equates to 100% light transmission. Plateletrich plasma stirred in the absence of platelet agonist equates to 0%light transmission (baseline, A). Change in platelet shape due toplatelet activation induced by addition of a platelet agonist results ina short decrease in the light transmission (B). When platelet aggregatesare forming an increase in the light transmission is recorded by thedevice and calculated as percentage of aggregation (C).

FIG. 6 shows the PRP storage containers: (a) 70 mL Nunc™ flask (smallestshown) (b) 30 mL Sterilin™ universal,

FIG. 7 shows Luckham R100 Rotatest Shaker

FIG. 8 shows the ELISA plate is provided already coated with a captureantibody directed against human PDGF-BB. The PDGF-BB present in testsamples or standards, the target protein, binds to the captureantibodies and becomes anchored to the plate. A second antibody directedagainst human PDGF-BB that is conjugated to Biotin is added which bindsto the PDGF-BB captured by the first antibody during a two hourincubation. Residual, unbound material is washed off and streptavidinconjugated to the enzyme horse radish peroxidise (HRP) is added to eachwell. Streptavidin forms strong, non-covalent 1:1 complexes with biotinduring a two hour incubation. Residual material is washed off and asubstrate for the enzyme, tetramethyl-benzidine, is added. The productof the enzyme-substrate reaction is coloured, the intensity of which isdirectly proportional to the amount of PDGF-BB in the samples, which isassessed against a standard curve. Diagram reproduced fromwww.mitosciences.com

FIG. 9 shows Collagen-induced aggregation: Day 1. Trace 1 and Trace 3:10 μg/mL collagen. Trace 2 and Trace 4: 5 μg/mL collagen,

FIG. 10 shows Collagen-induced aggregation for PRP stored in Nunc™flasks: Day 2, Trace 1 and Trace 2 Constant agitation MA: 27% Plt:257×109/L

-   Trace 3 and Trace 4 Intermittent agitation MA: 52% Plt: 241×109/L-   Trace 5 and Trace 6 Stationary MA: 65% Plt: 228×109/L-   Trace 7 and Trace 8 Constant agitation+PGE1 MA: 29% Plt: 243×109/L

FIG. 11 shows collagen-induced aggregation for PRP stored in universals:Day 2

-   Trace 1 and Trace 2 Constant agitation MA: 79% Plt: 149×109/L-   Trace 3 and Trace 4 Intermittent agitation MA: 92% Plt: 221×109/L-   Trace 5 and Trace 6 Stationary MA: 101% Plt: 215×109/L-   Trace 7 and Trace 8 Constant agitation+PGE1 MA: 77% Plt: 149×109/L

FIG. 12 shows collagen-induced aggregation for PRP stored in Nunc™flasks: Day 3, Trace 1 and Trace 2 Constant agitation MA: 19% Plt:245×109/L

-   Trace 3 and Trace 4 Intermittent agitation MA: 36% Plt: 202×109/L-   Trace 5 and Trace 6 Stationary MA: 40% Plt: 213×109/L-   Trace 7 and Trace 8 Constant agitation+PGE1 MA: 12% Plt: 221×109/L

FIG. 13 shows collagen-induced aggregation for PRP stored in universals:Day 3, Trace 1 and Trace 2 Constant agitation MA: 61% Plt: 128×109/L

-   Trace 3 and Trace 4 Intermittent agitation MA: 88% Plt: 221×109/L-   Trace 5 and Trace 6 Stationary MA: 97% Plt: 233×109/L-   Trace 7 and Trace 8 Constant agitation+PGE1 MA: 47% Plt: 145×109/L

FIG. 14 shows collagen-induced aggregation for PRP stored in Nunc™flasks: Day 4, Trace 1 and Trace 2 Constant agitation MA: 15% Plt:223×109/L

-   Trace 3 and Trace 4 Intermittent agitation MA: 23% Plt: 213×109/L-   Trace 5 and Trace 6 Stationary MA: 37% Plt: 209×109/L-   Trace 7 and Trace 8 Constant agitation+PGE1 MA: 15% Plt: 213×109/L

FIG. 15 shows collagen-induced aggregation for PRP stored in universals:Day 4, Trace 1 and Trace 2 Constant agitation MA: 43% Plt: 117×109/L

-   Trace 3 and Trace 4 Intermittent agitation MA: 65% Plt: 236×109/L-   Trace 5 and Trace 6 Stationary MA: 77% Plt: 243×109/L-   Trace 7 and Trace 8 Constant agitation+PGE1 MA: 34% Plt: 133×109/L

FIG. 16 shows collagen-induced aggregation for PRP stored in Nunc™flasks: Day 5, Trace 1 and Trace 2 Constant agitation MA: 13% Plt:205×109/L

-   Trace 3 and Trace 4 Intermittent agitation MA: 18% Plt: 193×109/L-   Trace 5 and Trace 6 Stationary MA: 30% Plt: 183×109/L-   Trace 7 and Trace 8 Constant agitation+PGE1 MA: 11% Plt: 200×109/L

FIG. 17 shows Collagen-induced aggregation for PRP stored in universals:Day 5, Trace 1 and Trace 2 Constant agitation MA: 61% Plt: 102×109/L

-   Trace 3 and Trace 4 Intermittent agitation MA: 64% Plt: 222×109/L-   Trace 5 and Trace 6 Stationary MA: 75% Plt: 233×109/L-   Trace 7 and Trace 8 Constant agitation+PGE1 MA: 27% Plt: 118×109/L

FIG. 18 shows Collagen-induced aggregation for PRP stored in Nunc™flasks: Day 8

-   Trace 1 and Trace 2 Constant agitation MA: 6% Plt: 191×109/L-   Trace 3 and Trace 4 Intermittent agitation MA: 12% Plt: 151×109/L-   Trace 5 and Trace 6 Stationary MA: 20% Plt: 146×109/L-   Trace 7 and Trace 8 Constant agitation+PGE1 MA: 6% Plt: 186×109/L

FIG. 19 shows Collagen-induced aggregation for PRP stored in universals:Day 8

-   Trace 1 and Trace 2 Constant agitation MA: 58% Plt: 85×109/L-   Trace 3 and Trace 4 Intermittent agitation MA: 39% Plt: 188×109/L-   Trace 5 and Trace 6 Stationary MA: 37% Plt: 208×109/L-   Trace 7 and Trace 8 Constant agitation+PGE1 MA: 27% Plt: 87×109/L

FIGS. 20 and 21 plot the changes in final percentage collagen-inducedaggregation over time in PRP variably stored in Nunc™ flasks anduniversal containers respectively

FIGS. 22 and 23 plot the changes in platelet count over time in PRPvariably stored in Nunc™ flasks and universal containers respectively

FIG. 24 shows Standard curve for PDGF-BB ELISA,

FIG. 25 shows Plots of supernatant PDGF-BB levels count over time invariably stored PRP FIGS. 25 and 26 plot the PDGF-BB levels insupernatants of collagen-activated PRP that had been variably stored inNunc™ flasks and universal containers respectively. Serial PDGF-BBlevels for supernatants of Nunc™ flask-stored PRP after collagenactivation. Note that a nominal level has been entered for theunavailable Day 2 result of constant agitation+PGE1 to permit fullgraphical plot

FIG. 26 shows Serial PDGF-BB levels for supernatants of universal-storedPRP after collagen activation. Note that a nominal level has beenentered for the unavailable Day 2 result of constant agitation to permitfull graphical plot.

FIG. 27 shows blood that has been separated by centrifugation. Startingfrom the bottom of each tube the first layer is Erythrocytes, the secondvery thin layer is of Leukocytes, above the leukocytes is the F2fraction of the plasma (also called PRP) (about 50% of the plasma) andright on the top is the F1 fraction of the plasma (also called PPP) (theother 50% of the plasma). It is the F2 (PRP) that was used in the testson platelet storage conditions that are described in this applicationand it is also the F2 (PRP) fraction that was used in the clinical testsdescribed in this application. F2 fraction is slightly darker than F1fraction which makes it possible for a practitioner to draw up the F1fraction first and then F2 fraction separately. The plasma fraction ismarked. The plasma fraction is separated into Fraction 1 (F1) (PPP) andFraction 2 (F2) (PRP). F2 is richer in platelets than F1 and thereforeF2 is preferably used in the methods of the present invention.

FIG. 28 shows blood that has been separated by centrifugation. Fraction1 of the plasma (F1) (also called PPP) is marked.

FIG. 29 shows blood that has been separated by centrifugation where theF1 fraction has been removed leaving the F2 fraction,

FIG. 30 shows 4 views, from top left clockwise: inside; front; back andfront open, of the centrifuge used to separate blood to provide F1 andF2 fractions of plasma. The centrifuge is a BTI-Endornet centrifugewhich spins the blood at 580 G for 8 minutes. During the centrifugationprocess blood gets separated into the fractions described in FIGS. 27 to29.

FIG. 31 shows a kit according to the present invention. The kitcomprises: a container that is sterile inside for keeping the plasma,PRP or F2 in, this is shown at the right hand side of the figure;several ampules containing the cosmetically acceptable composition,there may be one ampule for each application of the composition to theskin, the ampules are shown at the top of the figure; an agitatingdevice which can be used to constantly agitate the plasma, PRP or F2inside its container, this is shown at the bottom of the picture.

The cosmetic composition may be: Sun cream, After sun, Foundation,Tinted cream, tinted sun cream, Soothing anti redness cream with thegreen tint to mask redness of the skin, Scalp serum.

The Growth Factors:

For any PRP, 7 fundamental protein growth factors are concentrated inthe plasma after centrifuging (to what degree and with what level ofundesirable components will vary)

-   -   Platelet-derived growth factors—PDGF-AA, PDGFAB, PDGFBB    -   Transforming growth factors—TGFB1, TGFB2    -   Vascular endothelial growth factors—VEGF    -   Epithelial growth factors—EGF

This concentrate also contains 3 proteins in the blood to act as celladhesion molecules: fibrin, fibronectin, and vitronectin.

PDFG, VEGF and EGF are the most important for new tissue regeneration.The carrier may comprising one or more ingredients selected from table1.

The carrier may comprise

TABLE 1 All items stated include both their Organic and Syntheticversions. Item Function D522 (Dry Flo Plus) Absorbant D572 (Dry Flo AF)Absorbant K505 (Kaolin 2747) Absorbant M742 (Magnesium Carbonate)Absorbant S813 (Sorbosil BFG50) Absorbant C936 (Color Clay Rosa)Absorbant/abrasive A577-CC (Alistin P5) Active against cell membranedamage L592 (Latex LATZ) Adhesive polymer G513 (Glypure 70) AHA, pHadjuster L546 (Lactic Acid 90%) AHA, pH adjuster Z503 (Zincidone) Antidandruff A764 (Avenacare Beta Glucan) Anti oxidant D571-HM (DeepalinePVB) Anti wrinkle active M704-CC (Matrixyl 3000) Anti wrinkle activeS759 (Syn-tacks) Anti wrinkle active S761 (Syn-Ake) Anti wrinkle activeC911 (Symglucan) Anti-ageing active O581-CC (Oxygen Complex LS 9641)Anti-ageing active C642 (Cabosil M5) Anticaking agent V506 (VeegumUltra) Anticaking agent P564 (Protaderm HA) Antidandruff active S559(Sodium Salicylate) Antimicrobial S965 (Sodium Sulfite) AntimicrobialB507 (BHT) Antioxidant O510 (Oxynex K Liquid) Antioxidant P835 (PhytoTerra Organic Mate) Antioxidant S578 (Stay C50) Antioxidant V501(Vitamin E Acetate (dl-alpha Antioxidant Tocopheryl Acetate)) V528(Vitamin A Palmitate (1.7 iu)) Antioxidant V529 (Vitamin A Palmitate(1.0 ml U/g)) Antioxidant V550 (Ascorbyl Palmitate) Antioxidant V551(Tocopherols Mixed (Natural) INCI: Antioxidant Tocopherol) V552(dl-Alpha Tocopherol (INCI: Antioxidant Tocopherol)) v614 (D-AlphaTocopherol Natural) Antioxidant S717 (Sodium Disulfite Extra Pure)Antioxidant/preservative P822 (Phycosaccharide AIP)Antioxidant/protective extract M507 (Merquat Plus 3331) Antistatic agentP739 (Polyquarternium-7) Antistatic agent HEW574 (Actiphyte of WitchHazel PG) Astringent HEW575 (Actiphyte of Horse Chestnut Astringent/antiinflammatory PG) A589 (Organic Aloe Vera Powder 200:1 Botanical extractFreeze Dried) C582 (Codiavelane) Botanical extract C755-LM (CrodaromVelvet Flower) Botanical extract C786-AN (Cosflor Marshmallow HGL-1Botanical extract (PS)) C787-AN (Cosflor Mango HGL-1 (PS)) Botanicalextract E560 (Emblica) Botanical extract HEO503 (Green Tea Lipo S)Botanical extract HEO596 (Organix Marigold PS) Botanical extract HEW1040(Witch Hazel Organic) Botanical extract HEW1066-MB (Green Tea LG)Botanical extract HEW1085-MB (Ceapro Oat Botanical extractAvenanthramides) HEW1103-MB (Witch Hazel Distilled, Botanical extractPhenoxyethanol) HEW1122 (Specifix Chamomile FG) Botanical extractHEW1130 (Specifix Yarrow 1579) Botanical extract HEW1136 (SpecifixComfrey Leaf PF) Botanical extract HEW1154 (Orange Secrets) Botanicalextract HEW503 (Extrait Concombre HG) Botanical extract HEW505 (AloeVera 10:1) Botanical extract HEW596 (NAB Fennel Seed Extract) Botanicalextract HEW643-LL (Witch Hazel) Botanical extract HEW717 (Extract NABRed Clover Botanical extract Isoflavones) HEW822 (Green Tea ECO Extract)Botanical extract HEW863 (Rosewater) Botanical extract HEW966 (CalendulaECO) Botanical extract HEW987 (Cosflor Roman Chamomile Botanical extractHGL-1) P850-CC (Pronalen Ruscus Spe) Botanical extract P909 (ProteasylLS8951 PW) Botanical extract R516 (Regu-SEB (331-01)) Botanical extractR563 (Rosamine R) Botanical extract R566-CC (Remoduline B E1) Botanicalextract S981-AN (Slippery Elm Bark Powder) Botanical extract D642(Dissolvine GL-38) Chelating agent N502 (Dissolvine Na2 (EDTA Na2))Chelating agent N504 (Dissolvine NA (tetra sodium)) Chelating agentP666-HM (Procircul 8) Circulation active A611 (AC Yeast Beta Glucan)Collagen producing active Z507 (Zinc Oxide EP) colouring agent C766(Claritea Prov) Dark circle/complexion lightening active S586 (SalicylicAcid) Denaturant P831 (Proteasyl TP POE LS 9818) Elasticity active B515(Butylene Glycol) Emolient B586 (Cocoa Butter (Blanova)) Emolient C534(Cetiol OE) Emolient C581 (Cetiol V) Emolient C608 (Organic CupuacuButter Refined Emolient Grade) C673 (Cocoa Butter) Emolient C760 (CetiolC5) Emolient C918 (Cetiol AB) Emolient D585 (Dragoxat 89) Emolient D783(DC245) Emolient G523 (Glucamate LT) Emolient G549 (Glucamate SSE20)Emolient G550 (Glucate SS) Emolient H568 (Heliogel) Emolient H592-HM(Hexyl Laurate) Emolient H593 (Hydrogenated Polyisobutene) EmolientHEO502 (Aloe Vera Oil Extract CG) Emolient I512 (Isopropyl Palmitate)Emolient I513 (Isopropyl Myristate) Emolient I564 (Isopropyl Myristate)Emolient I569 (Isononyl Isononanoate (Dubb ININ)) Emolient I570(Isopropyl Isostearate) Emolient L548 (Lipovol MOS-70) Emolient L595(Linosa PEG-7 Glyceryl Cocoate) Emolient L642 (Lipex Shea) Emolient L645(Lipex Sheasoft) Emolient M726 (Mikrokil Cos (12002)) Emolient OIL503(Almond Oil (Sweet) - Refined) Emolient OIL506 (Jojoba Oil Light)Emolient OIL511 (Grapeseed Oil) Emolient OIL512 (Avocado Oil) EmolientOIL520 (Peach Kernel Oil) Emolient OIL522 (Soybean Oil) Emolient OIL533(Blackcurrant Seed Oil) Emolient OIL536 (Olive Oil - Refined) EmolientOIL541-AN (Castor Oil, Refined) Emolient OIL568 (Organic Jojoba Oil)Emolient OIL572 (Organic Coconut Oil) Emolient OIL574-AN (Hemp Oil,Refined) Emolient OIL590 (Organic Grapeseed Oil) Emolient OIL593(Calendula Oil 1210) Emolient OIL599 (Organic Shea Butter) EmolientOIL606 (Organic Sweet Almond Oil) Emolient OIL613 (Organic EveningPrimrose Oil) Emolient OIL614 (Organic Wheatgerm Oil) Emolient OIL619(Camelina Oil Refined) Emolient OIL621 (Organic Almond Oil) EmolientOIL633 (Coconut Oil - Fractionated Emolient (liquid)) OIL638 (RapeseedOil Refined) Emolient OIL655 (Coconut Oil RD) Emolient OIL658 (HerbalExtract Aloe Vera Oily) Emolient OIL659 (Sunflower Oil Refined) EmolientOIL661 (Jojoba Oil, Golden) Emolient OIL669 (Mineral Oil BO) EmolientOIL676 (Tea Tree Oil Organic) Emolient OIL678 (Olive Oil, Organic)Emolient OIL686 (Avocado Oil Refined) Emolient OIL706 (Organic CastorOil) Emolient OIL722 (Macadamia Oil Organic) Emolient OIL729-NK (TamanuOil, stabilised) Emolient OIL734-CC (Corn Oil Refined) Emolient OIL751(Hazlenut Oil, Refined) Emolient OIL756 (Rosehip Oil, Refined) EmolientOIL809 (Organic Safflower Oil) Emolient OIL830 (Soyabean Oil Refined)Emolient OIL831 (Lady's Thistle Oil) Emolient OIL841 (Meadowfoam SeetOil) Emolient OIL853 (Avocado Oil, Crude) Emolient OIL867-AN (BabobabOil Organic) Emolient OIL870 (Calendula Oil) Emolient OIL907 (OrganicDeodorised Argan Oil) Emolient S1014 (Silicone CM56 (DC345)) EmolientS1050 (Saboderm ISN) Emolient S510 (Stearyl Alcohol) Emolient S523(Silicone DC 200 Fluid 350 CST (see Emolient B665)) S574 (Silicone DC200/100) Emolient S575 (Silicone DC 1501) Emolient S614 (Silicone DC245) Emolient V588-AN (Viatenza Shea PO6) Emolient W516 (Wickenol 156(replaces L503)) Emolient T527 (Tegosoft LSE 65K Soft) Emolient/foamenhancer/viscosity modifier A504 (Amphisol K) Emulsifier A633-AN (Axol C62 Pellets) Emulsifier C736 (Span 60-PA-(SG) (previoulsy CrillEmulsifier 3 - sorbitan stearate)) C750-CC (Crovol A70 UK) EmulsifierC813 (Crodafos CES) Emulsifier C815 (Ceteareth 25) Emulsifier C933-CC(Cetyl Palmitate 95%) Emulsifier D609 (Dermofeel SL) Emulsifier E529(Euperlan PK 1200) Emulsifier E633 (Edenor C12 98-100 MY) EmulsifierK570 (Kemest GDS) Emulsifier K571 (Kemionic SLES 228) Emulsifier L536(Lexemul 561) Emulsifier L583 (Lexemul T) same as G621) Emulsifier L601(Linosa SLES 70) Emulsifier M519 (Montanov 68) Emulsifier M550 (Montanov202) Emulsifier M564 (Montanov S) Emulsifier P559 (Procol CS-20D)Emulsifier P680 (Procol CS 20 (Ceteareth-20)) Emulsifier P869-MB(Palmitic Acid) Emulsifier S748 (Surfac JH 200) Emulsifier S840(Surfhope SE Cosme C-1616) Emulsifier T609 (Tegosoft PC41) EmulsifierS677 (Surfac UN90) Emulsifier/degreasing agent H554-SD (Hydramol TGLEster) Emulsifier/skin conditioning agent S976 (Sveltine LDRM 961S)Emulsifier/skin conditioning agent S718 (Sorbitan Mono Stearate)Emulsifier/surfactant C810 (Surface CS (Cetearyl Alcohol)) Emulsioinstabiliser B648 (Organic Beeswax) Emulsion stabiliser B661 (Beeswax,white granules (8104)) Emulsion stabiliser C674 (Cetyl Alcohol) Emulsionstabiliser E566 (Emulium Kappa) Emulsion stabiliser M606 (Montanov L)Emulsion stabiliser S1003 (Stearic Acid) Emulsion stabiliser S505(Stearic Acid 1810) Emulsion stabiliser HEW602 (NAB Willow Bark Extract)Exfoliating BHA E530 (Peeling Enzymatique) Exfoliator F561 (Florabeads28/60 Arizona Sky) Exfoliator P521 (Pumice Powder Coarse (50 mesh))Exfoliator T614 (Tabishirex) Exfoliator A607 (Antileukine 6) Extractwith UV protection HEW672 (NAB Arnica Extract) Eye active(inflammation/puffiness) L606 (Liponate ISA) Fatty acid L565 (LuvisetClear) Film former L727 (Luviskol K30) Film former P657 (Pepha-Tight)Film former D683 (Daitosol 5500GM K-5136) Film former C759 (Covacryl SP)Film former/conditioning agent C762-SD (Copolymer 845 G) Filmformer/conditioning agent E531 (Egg White Powder P11) Filmformer/conditioning agent L713 (Luviquat UltraCare) Film former/hairfixative M663 (Mirustyle XHP) Film former/hair fixative B643 (BiopeptideEL) Firming active T682-CC (Tensine) Firming active T685-CC (Tensine 2)Firming active HEW671 (Extract NAB Mushroom) Firmness and anti wrinkleactive S696 (Sodium Bicarbonate) Fizzing agent A640 (GLB60 (Antil HS 60)Foam booster/viscosity modifier D625 (DC949 Cationic Emulsion) Hairconditioning agent M644 (Mirustyle MFP PE) Hair conditioning agentS979-SD (Styleze CC10) Hair conditioning agent T666 (Tinocare SiA1) Hairconditioning agent V603-AN (Varisoft 432 CG) Hair conditioning agentV606-AN (Varisoft BTMS Flake) Hair conditioning agent K558 (Keratec IFPPE) Hair/skin conditioning agent G537 (Glycerin (Surfac G995V))Humectant G540 (Organic Glycerine) Humectant G628 (Organic Glycerin)Humectant HEW585 (Extrait Ginseng HG) Humectant HEW677 (Extract NABSiberian Ginseng) Humectant L549 (Lubragel Oil) Humectant L556(Lactofill Sensitive) Humectant L622-CC (Lubrajel PF) Humectant L717(Lubrajel Oil Free) Humectant P501 (Propylene Glycol) Humectant P553(D-Panthenol 751 (liquid)) Humectant P668 (Protachem GL26 (same asL616)) Humectant S521 (Squalane - Olive Derived (was Humectant D527))S522 (Sodium Hyaluronate 1% Solution Humectant (Liquid)) S545 (SorbitolSyrup 70% (Sorbidex NC Humectant 16205)) S771 (Sodium Hyaluronate Salt(Powder)) Humectant W503 (Waglinol 6014 (IPM)) Imolient S676 (SolaveilCT-100 Clarus) Inorganic sunscreen Z502 (Zinc Oxide CM3K 50 XZA)Inorganic sunscreen S789 (Suberlift) Lift active N600-CC (Nano LPDsArbutin PF) Lightening active S952 (Surfac HT10) Low foam surfactantB610 (Bamboo Exfoliator 500) Micro exfoliator D545 (Diamond Powder SY-FS60/70) Micro exfoliator P601 (Pearl Powder 125) Micro exfoliator R557(Rice Exfoliator 500) Micro exfoliator I561 (Isocell MAP)Microencapsulated antioxidant U532-CC (UV Titan M 262) Mineral/inorganicsunscreen N598-CC (Norgel) Moisturiser H594-CC (Hydrosoy 2000 PE)Moisturising L547 (Lamesoft PO65) Moisturising V534-CC (Viamerine 4000)Moisturising T557 (Tagravit F1) Moisturising/anti-ageing K562 (KahlBerry Wax 6290) Natural wax, texturiser A562 (Acusol OP 301) OpacifierM727 (Myristic Acid PC) Opacifier T687 (Tego Pearl N300) Pearliser C510(Citric Acid Monohydrate) pH adjuster P886 (Potassium Hydroxide) pHadjuster S517 (Sodium Hydroxide) pH adjuster S960 (Sodium Hydroxide 45%solution) pH adjuster T501 (Triethanolamine 99% (T.E.A)) pH adjusterC536 (Calcium Carbonate) pH buffer T691 (Tris Amino Ultra PC) pH bufferG569 (Gelinnov) Polymer L715 (Luvigel EM) Polymer/skin conditioningagent D589 (Dermosoft Octiol) Preservative G609 (Geogard 221)Preservative P590 (Potassium Sorbate Granules 105119) Preservative PR502(Nipagin M) Preservative PR504 (Kathon CG) Preservative PR505 (NipasolM) Preservative PR506 (Nipastat) Preservative PR508 (Phenonip)Preservative PR510 (Phenoxetol (Phenoxyethanol) use Preservative PR572)PR515 (Germaben II) Preservative PR518 (Euxyl K100) Preservative PR526(Euxyl K702) Preservative PR533-CC (Paratexin CPS Preservative(Chlorphenesin)) PR546 (Germall 115 USP (Powder)) Preservative PR547(Euxyl PE 9010) Preservative PR548 (Grapefruit Seed Extract G2)Preservative PR561 (Optiphen) Preservative PR563 (Benzyl Alcohol)Preservative PR568 (Sodium Benzoate) Preservative PR572 (Saliethanol(phenoxyethanol)) Preservative PR580 (Geogard Ultra) Preservative PR585(Sorbic Acid) Preservative PR588-LM (Euxyl K712) Preservative PR593(Euxyl K701) Preservative PR595 (Elestab CPN) Preservative PR607(BlagGuard GPL (same as PR537)) Preservative S963-CC (Symdiol 68T(177441)) Preservative booster/antioxidant S950 (Sensiva SC 10)Preservative/emolient HEW675 (Actiphyte of White Tea GL) Protective I546(Protanal FM 6130 (Isagel FM Setting agent Alginate)) A774 (AvenacareECO Oat Beta Glucan) Skin and hair rejuvenation HEW963 (OligophycocorailSPE) Skin balancing A734 (Aloe Vera Gel Base) Skin calming HEW885(Horsetail Extract (1552)) Skin conditioning HEW896 (Camomile ECO) SkinConditioning A502 (Allantoin 98.9%) Skin Conditioning Agent A676-AN(Aquarich) Skin conditioning agent A732-CC (AC Oak Kernel ProteinPowder) Skin conditioning agent B503 (Bernel Ester TCC) Skinconditioning agent C577 (Cosmocair C100) Skin conditioning agent C698(Cytobiol Lumin Eye) Skin conditioning agent C949-CC (Cetiol S) Skinconditioning agent C954 (Cegesoft C24 (same as T526)) Skin conditioningagent D564 (DC556) Skin conditioning agent D654-AN (Dermofeel P-30) Skinconditioning agent F509 (Fucogel 1000PP) Skin conditioning agent F541(Fucogel 1.5P) Skin conditioning agent G563 (Gatuline Age Defence 2)Skin Conditioning agent G589-AN (Gluadin Wlm Benz) Skin Conditioningagent G605 (Gatuline Skin Repair Bio) Skin conditioning agent G631(Gatuline RP) Skin conditioning agent I560 (Isododecane (CSI Code 5108))Skin conditioning agent K567-AN (Kapilarine) Skin conditioning agentM543 (Mango Butter Refined) Skin conditioning agent M625 (Myritol 312)Skin Conditioning agent P556 (Peg 8) Skin conditioning agent P658(Pentavitin) Skin Conditioning agent S804 (Shea Butter, Refined) SkinConditioning agent S825-CC (DC2501 Cosmetic Wax) Skin conditioning agentS982-AN (Sunflohair) Skin Conditioning agent U502 (Urea) Skinconditioning agent V509 (Vitamin F Glycerinester O/S) Skin conditioningagent V546 (Vitamin A (Water Miscible) Type Skin conditioning agent 100)A629-CC (AC Zaatt) Skin conditioning and lightening agent A653(Achromaxyl IS) Skin lightening agent B677 (Belides ORG) Skin lighteningagent P779 (Phytexcell Mulberry) Skin lightening extract D647 (DismutinJ PF) Skin protecting antioxidant S1010 (Schercemol 1688 Ester)Skin/hair conditioning agent T709 (Triethylhexanoin) Skin/hairconditioning agent P839 (Peptan SR Marine) Skin/hair conditioning gentD523-LL (Solubiliser 660352) Solubiliser N608 (Natragem S140NP)Solubiliser P519 (Polysorbate 60 (Protasorb S-20 NF)) Solubiliser P587(Protasorb O-20) Solubiliser P622-HM (Procol OA-20) Solubiliser P646(Protachem HCO 40) Solubiliser P880 (PEG-20 Glyceryl Laurate (TagatSolubiliser L2)) S535 (Surfacare T20) Solubiliser S564 (SolubilisantLRI) Solubiliser C951 (Crillet 3 (Tween 60-SS)) Solubiliser/emulsifierC719-CC (Cibafast H Liquid) Solvent E504 (Ethanol DEB 100) Solvent T619(Transcutol CG) Solvent HEW980-CC (Cosflor Cucumber HGL-1 Soothing (PS))P823-HM (Phytexcell Centaury) Soothing/astringent extract A604(Aquacacteen) Soothing/calming/firming extract HEW673 (Authenticals ofCucumber) Soothing/calming/firming extract S823 (Syntran PC5227CG) SPFbooster HEW625 (Actiphyte of Paraguay Tea SPF booster/irritancy reducerof AHA's Conc.) M735 (MALTODEXTRIN (replaces D529)) Stabiliser O564-LL(Oxynex ST Liquid) Sunscreen stabiliser A542 (Steol CA-330-E (AES 136))Surfactant C777-CC (Crodasinic LS 30) Surfactant D566 (Dehyton K(Sabosol PB)) Surfactant H523 (Hostapon SCI 85 G (Granular)) SurfactantH540 (Hostapon CT TEIG) Surfactant K569 (Kemthox FA S 21 (P776))Surfactant O523 (Oramix CG 110 (Sue Stowell)) Surfactant O532 (Oramix NS10) Surfactant P527 (Plantacare 2000 UP (Kemgluco Surfactant CEHL))P534-AN (Plantacare 818) Surfactant P538 (Kemgluc CLM Plantacare 1200UP) Surfactant P879 (Procetyl AWS) Surfactant S1047 (Steol CS-330)Surfactant S547-LM (Surfac GMS NSE40) Surfactant S710 (Stepanol AM 30)Surfactant T512 (Tegobetaine F50) Surfactant V599 (Varisoft 300)Surfactant S766 (Steol BES70 D5) Surfactant/emulsifier S777 (SucragelCF) Surfactant/emulsifier V511 (Varisoft BT85 (Pellets)) Surfactant/hairconditioning agent S795 (Surfacare DHA) Tanning agent E576-HM(Erythrulose) Tanning enhancer C920 (Covabead PMMA) Texturising effectB673 (BRG SG 116 (DC 9040) Texturising Silicone C503 (Carbopol Ultrez10) Thickener X507 (Xperse 201) UV broad spectrum protection S699(Sunspheres Powder) UV protection T683 (Tinosorb M) UVA absorber UV504(Uvinul MS40 (Benzophenone-4)) UVA absorber UV505 (Parsol 1789) UVAabsorber UV514 (Uvinul A Plus Granular) UVA absorber Z505 (Z-Cote HP1)UVA and UVB protection UV510 (Eusolex 4360 (Benzophenone-3)) UVA/Babsorber E571 (Eusolex t-2000) UVB absorber UV502 (Parsol MCX) UVBabsorber UV525-CC (Escalol 587) UVB absorber C955 (Carbopol 980)Viscosity control L721 (Lexfilm Sun) Viscosity control M565 (Mackol CAS100N) Viscosity control V527-CC (Viscarin GP209NF) Viscosity controlA539 (Aculyn 28) Viscosity modifier A601 (Amigel) Viscosity modifierC584 (Carbopol Ultrez 21) Viscosity modifier C594 (Carbopol Ultrez 20)Viscosity modifier H555-HM (Wacker HDK H20) Viscosity Modifier K502(Keltrol F) Viscosity Modifier K531 (Keltrol CG TE) Viscosity ModifierK532 (Keltrol CG-RD) Viscosity Modifier K537 (Keltrol CG SFT) ViscosityModifier L651 (Lanette D) Viscosity modifier M528 (Methocel J75 MS)Viscosity Modifier N509 (Natrosol 250 HHR) Viscosity Modifier N533(Natrosol Plus CS Grade 330) Viscosity Modifier P530 (Pemulen TR 2)Viscosity Modifier S589 (Structure XL) Viscosity modifier S723 (SimulgelNS) Viscosity modifier A554-HM (Antil 171) Viscosity modifier forsurfactants S544 (Sepigel 305 × 4) Viscosity modifier/emulsifier A766(Avicel PC-611) Viscosity modifiers S1036 (Sylvaclear A200V)Waterproofing S501 (Sodium Chloride (salt)) C905 (Organic Cocoa Butter)Collagen Ingredients Marine Hydrolysed Collagen LMW Hydrolysed Collagen(Marine) Collagen 1% Soluble collagen (porcine) Collagen HydrolysateCosmetic N-SS Hydrolysed Collagen Solu-Coll Native Soluble Collagen(Cattle) Solu-Coll M Soluble Collagen (Marine) Solu-Mar Native SolubleCollagen (Marine) Dermosot 1388 Preservative Retinol (tretinoin)Preservative Saponin Preservative Q Enzyme 10 PreservativeGlycerrhinitic Acid Preservative Fragrances FRAG1065 (Fresh StylePN994475) FRAG1104 (Celebrity Chic UKB06022) EO806 (Organic Tea TreeOil) EO807 (Organic Lavender Oil) EO898 (Orange Sweet Essential OilUN1169)

TABLE 2 Caprilyc capric triglyceride, Stearic acid, Glyceryl Strearate,Cetearyl Alcohol, Q10, sodium Lauroyl, Glutamate, Retinol Palmitate,Cetearyl Alcohol, Shea butter, Isolanolin, Sorbitan stearate, lactate,urea, Hyluronic Acid, Saponins, D-Panthenol, Glycerin, Sodium Anisate,sodium levulinate, Glyceril Caprilate, EDTA, CaprylyL Glycol, PotassiumSorbate, Caprylic acid combo, Cetyl palmitate, Stearic acid, Cetearylalcohol, Sodium lauroyl glutamate, Caprilyc capric triglyceride,Sorbitol, Glyceryl stearate, Demin sterile water, Disodium EDTA 0.1%,Hyluronic acid, Retinol (tretinoin) 0.5%, Saponin, Q enzyme10,Glycerrhinitic acid, Collagen, lacto ceramide, L-Cartinine, Lactic acid,Glycolic acid, Sorbitan stearate, Sorbitol, Lactate, Decandenol,Licorice extract, Green tea Extract, Arnica, calcium chloride,gluconate, Thrombin, Demineralised sterile water, Vitamin A, PEG-40sterarate, Ascorbyl palmitate, Glycine Argenine HCL, Sodium hyluronate,Lauroyl Lysine

TABLE 3 1 - Bladderwrack extract (seaweed): Derived from the driedthallus (bulbous root) of Fucus vesiculosus, a type of seaweed. 2 -Horsetail extract: Equisetum arvense, commonly known as horsetail,mare's tail, shave grass, or bottle brush, is a plant that growsthroughout central Europe. 3 - Hydrocotyl extract: Hydrocotyl asiatica,commonly known as gotu kola or Indian pennywort.

The composition may comprise one or more natural ingredients listed intable 3.

TABLE 4 EDTA Glycerin Xanthan Gum Avenacare Beta Glucan - Beta Glucan,Water, Maltodextrin Collasurge - Aqua, Collagen Amino Acids, PotassiumSorbate, Ethylhexyl Glycerin, Phenoxyethanol Crotein M - HydrolysedCollagen Sodium Hydroxide

TABLE 5 Preservatives: Dermosoft 1388 - Water, Levulinic Acid, Parfum,p-Anisic Acid, Sodium Hydroxide, Glycerin Gerogard 221 - Benzyl alcohol,Water, Dehydroacetic Acid Dermosoft GMCY - Glyceryl Caprylate Potassiumsorbate

FIG. 1 shows a side view of an embodiment of a container according tothe present invention. The container 10 may be made of any suitablematerial, for example rigid or flexible plastics.

Alternatively, one of the chambers, 11 may be arranged to be filled withan activator 12 a according to the present invention. One of thechambers 13 may be filled with a cosmetically acceptable carrier 19 andmay be arranged with an opening, valve or port 18 arranged to allowplatelet rich plasma (PRP) to be introduced into the chamber so that itmixes with the cosmetically acceptable carrier. The opening, valve orport may be arranged to be re-sealable after introduction of the PRP,for example by being equipped with a lock or cap or being a rubber sealthat can be injected through.

The container may further comprise a mixing chamber 14 in communicationwith each of the chambers 11 and 13 so arranged that contents of each ofthe chambers can enter the mixing chamber in a required ratio. A valve,seal, cap or lock may close an exit from the mixing chamber 17 whichallows the contents of the mixing chamber to exit the container whenrequired. The container may be activated, for example by squeezing thecontainer or by a mechanism that urges the contents of the two chambers11 and 13 into the mixing chamber 14 in a required ratio and the mixedcontents out of the container via an exit 17. The container may beactivated on multiple occasions, each activation causing a suitableportion of the contents of each of the chambers to be mixed together andto exit the container.

FIG. 2a shows a view of an embodiment of an embodiment of the containerof the present invention. The container comprises a chamber 20 suitablefor containing PRP, the container 20 may be sterile. The containercomprises a multiplicity of chambers 21 comprising a cosmeticallyacceptable carrier 22. The cosmetically acceptable carrier may comprisean activator. The chambers comprising cosmetically acceptable carriercan be opened and a suitable amount of PRP can be added to thecosmetically acceptable carrier and mixed before applying thecomposition to the skin.

FIG. 2b shows a side view of a chamber suitable for containing PRP. Thechamber 23 has a top lid 24 and a bottom lid 25. FIG. 2c shows thechamber 23 with the top lid removed to show that the chamber is equippedwith a dropper 26 to allow drops of PRP to be shaken out of the chamber.FIG. 2d shows a view of the chamber 23 upside down with the top lid 24in place. The bottom lid has been removed. PRP in a syringe 27 with aneedle, 28 is injected through a rubber seal 29 that allows PRP to betransferred into the chamber under sterile conditions.

Tests on Human Volunteers

Platelet Rich Plasma is to be used topically, in combination with aSerum containing Collagen. The Collagen in the serum, when in contactwith the PRP, induces platelet activation and release growth factorsfrom the activated platelets.

A serum containing collagen has been prepared with the followingformulation:

TABLE 6 CAS Number INCI Function/Description % 7732-18-5 Aqua (Water)Solvent 94.5895 56-81-5 Glycerin Humectant & Moisturiser 2.001573049-73-7 Hydrolyzed Collagen Skin Conditioning Agent 1.0600 26402-26-6Glyceryl Caprylate Emollient, Emulsifier, Emulsifier: HLB 06- 1.000010.9 11138-66-2 Xanthan Gum Emulsion Stabiliser, Skin Conditioning0.9000 Agent, Surfactant, Viscosity Control 110-44-1 Sorbic AcidAntimicrobial 0.3000 139-33-3 Disodium EDTA Chelating Agent 0.10009050-36-6 Maltodextrin Absorbant &/or Abrasive Powder, 0.0200 EmulsionStabiliser, Film Former &/or Hair Fixative 532-32-1 Sodium BenzoateAntimicrobial 0.0100 Oat Beta Glucan Skin Conditioning 0.0100 24634-61-5590-00-1 Potassium Sorbate Antimicrobial, Vitamin and vitamin 0.0060derivatives 123-76-2 Levulinic Acid Skin conditioning 0.0015 19856-23-6Sodium Levulinate Skin conditioning 0.0015 100.0000

Product Specification

The Collagen Serum is a pale yellow, thin liquid with a hazy appearance.

The pH of the Collagen Serum is pH 5.20-5.50 and the Viscosity is4000-7000 cps using Spindle 4@5.

Product Stability and Preservation

The Collagen Serum remains suitably stable for the duration of astability test, with a recommended shelf life determined to be 12 monthsfrom the date of opening.

The Collagen Serum has passed a full challenge test carried out to theEuropean Pharmacopoeia standards.

Clinical Tests

9 subjects were recruited who wanted to improve the appearance of areasof their skin. These patients were divided into three groups.

Group 1

Blood was taken from each group 1 subject and centrifuged to separateplasma from other blood components such as red blood cells and whiteblood cells. Only the richest part of plasma called PRP or F2 fractionwas collected as this is the fraction that is richest in platelets. Thepoorer fraction: PPP or F1 was not used in the serum.

PRP was injected into the skin using a series of intradermal injectionsacross the treated area. The remaining PRP was stored. Each day for thefollowing eight days a sample of PRP was mixed with equal volume ofserum containing collagen and the mixture was applied to the treatedarea. On day eight further blood was taken and fresh PRP F2 wascollected so that the topical treatment was continued for another 8 daysafter the day of injection treatment.

Group 2

Blood was taken from each group 2 subject and centrifuged to separateplasma from other blood components such as red blood cells and whiteblood cells. Only the richest part of the plasma, PRP fraction or F2 wasseparated and collected from the rest of the plasma as this is thefraction that is richest in platelets.

The PRP was stored. Each day for the following eight days a sample ofPRP was mixed with equal volume of serum containing collagen and themixture was applied to the treated area on one side of the subject'sface. On day 8 and 16 further blood was taken and fresh PRP (or F2) wascollected so that the topical treatment was continued for a total of 24days after the start of treatment treatment. No PRP injections weregiven to this group, only topical treatment with PRP mixed with collagenserum.

Group 3

The subjects in group 2 applied the collagen serum twice a day for 24days without any injections or any PRP mixed with the serum.

Results

The appearance of the skin on the treated area before and after thetreatment for each subject is shown in FIG. 4. Pictures of the treatedareas of patients 1, 2 and 3 from group 1 and patients 1, 2 and 3 fromgroup 2 show a comparison of the skin before the treatment and after thetreatment. Group 1 had PRP injections in the treated area followed byusing serum mixed with PRP each day for 16 days. Group 2—only used serummixed with PRP each day for 24 days on one side of their face so wecould compare the difference with the other side of their face Group3—used the collagen serum only (without adding PRP to it).

After using the PRP Injections for as anti aging treatment we know thatPRP activates tissue regeneration and improves the tone and skinelasticity.

By:

-   stimulating the natural production of Hyaluronic Acid by our own    cells    -   promoting the increased secretion of Collagen and Elastin which        generates a greater consistency, firmness and reduced sagging in        the skin    -   it improves the skin hydration-   softens lines and wrinkles    -   it increases the luminosity and gives the skin a higher luster.

The only problem with the injections is the post operative swelling,bruising caused by many injections all over the sensitive areas likeface and neck, soreness and the fact that the effect doesn't last verylong which is why the present invention will dramatically improve theeffect of the PRP, here dude the recovery time from the post operativeproblems and maintain the results for much longer. The only problem withthe injections is the post operative swelling, bruising caused by manyinjections all over the sensitive areas like face and neck, soreness andthe fact that the effect doesn't last very long. The advantage of thepresent invention is that mixing PRP with serum and using it post PRPinjections dramatically improves and prolongs the effect of the PRP andalso allows the active factors to penetrate into the skin without theneed for injection. The PRP with serum can be used as a course oftreatment where a portion of PRP is mixed with a portion of serum eachday to activate fresh platelets every day and the activated plateletsare applied to the skin. This treatment can be carried out alone orafter a course of treatment of PRP injection to prolong and enhance theeffects of the PRP by injection treatment and also to help reduce theamount of bruising, swelling and soreness caused by the PRP by injectiontreatment.

Feed Back from the Clients in Group 1, 2 and 3

Group 1

Client 1: was a 47 years old lady who had PRP injections both in theface and hands followed by using our plasma serum for 8 days. Before thetreatment she suffered from red patchy areas on her cheeks and the sideof her nose as you can see in the first picture below on the left.Immediately after the PRP injections her face was slightly swollen andinflamed because of the injections. It also felt very sore. I made herthe plasma serum that day and she started using it immediately andcarried on for 8 days.

After 8 days the red patchy areas were all gone. The dark circles underthe eyes improved. She reported that her skin felt softer, plumper andmore hydrated and she had a healthy “Glow”. She no longer needed to useany foundation on her skin.

The same client had her hands treated by PRP injections followed by 8days using the plasma serum

Hands Before:

Skin was very dry. Red patchy areas and deep lines around the wrist.Even using hand creams several times daily was not enough to keep theskin moisturized and soft. As a result skin looked old and wrinkly

Hands after Treatment

The skin was much softer, tighter and more hydrated. The redness hadfaded dramatically on the wrist and the deep lines had softened.

The soreness and bruising after the PRP injections healed very quickly,much quicker than it would normally

The skin on the hands felt soft and was glowing. It was softer, morehydrated, lines and patchy red areas had faded dramatically

Client 2: This was a 67 years old lady with deep frown lines and lots ofsmaller lines around the eyes, on the cheeks and the chin. She had deepdark circles below the eyes and patchy red/brown areas all over herface. To this lady sagging was the biggest problem and previous fillertreatments on her face weren't giving her the result she desired.

She was treated with just PRP injections 9 months ago. She took about 5days to recover from post-operative swelling and bruising. After thatskin felt great but it didn't last very long.

This time I treated her with PRP injections first followed by using myPlasma serum for 8 days.

Not only she recovered from post-operative swelling, soreness andbruising within 24 hours, her skin continued to feel softer, plumper,and brighter and had a very healthy glow. The biggest improvement withthis lady was the tightening of the skin. She described it as ‘A gentleface lift’

The little lines on the cheeks and chin softened dramatically. The deepfrown lines also softened. The areas under her eyes looked brighter andhealthier and her skin had a more even and brighter tone.

Client 3 This was a 32 years old man with a very deep Nasio Labial fold(laughter line) which made him look older than his age. The skin insidethe fold was also very dry and made shaving very hard for him.

This subject was treated with one session of deep PRP injectionsfollowed by using the Plasma serum for 8 days. The fold didn'tcompletely disappear but improved a lot, it now looks more like a fineline. The skin also felt softer, tighter and more hydrated.

Group 2:

The clients in this group reported very similar feedback: The treatedside of the face felt much tighter, more hydrated, plumper and smootherthan the untreated side throughout the whole 24 days despite being outin the sun and wind.

The dark circle around the eyes looked much lighter.

They all reported a new healthy “Glow”

As a result of this rehydration the lines around the eyes looked muchsofter.

Those who had very dry and patchy skin and after using the serum for 24days reported softer, plumper, tighter and more even tone on the skin onthe treated side while the untreated side remained the same.

GROUP 3: This Group Reported No Significant or Visible ImprovementsOther than the skin felt a little more moisturized whenever they appliedthe collagen serum on but the effect didn't last more than 2-3 hours.

Kit

The kit may contain:

Eight plastic capsules containing the collagen serum (cosmeticallyacceptable carrier comprising collagen activator) for 8 days use or asuitable number of plastic capsules, one for each day or one for eachapplication of the PRP. The amount of collagen serum in each capsule maybe enough for one application. The collagen serum will be mixed with thesame volume of PRP, mixed between finger tips and applied on the face,neck, chest and/or hands.

A container for holding the PRP made of a suitable material, for exampleSteriline polypropylene which according to the test described in thisapplication has proven to be the best material for storage of PRP (alsocalled F2 which is the richer part of plasma).

The PRP container may be designed to reduce the amount of air that flowsinto the bottle so that there is some oxygen inside but not a constantflow of oxygen. The container may be arranged to dispense the PRP in adropwise manner so that the user can dispense a suitable number of dropsof PRP to be mixed with the cosmetically acceptable carrier for eachapplication.

3— A small battery driven vibrating table is also included now so thatthe client can keep the PRP agitated for the 8 days. Although our testshowed great results even with the PRP group that was only shaken a fewtimes/day we know that the best results are always achieved if the PRPis agitated constantly which is why a little vibrating table is in thekit.

Platelet Storage Conditions

Whilst PRP is generally preferred to recombinant growth factors inpromotion of wound healing there is a paucity of data on stability ofPRP preparations in this clinical setting. Being anucleate, plateletshave a short in vivo lifespan of 8-10 days, and platelet donations takeninto citrate-based anticoagulants stored at 22° C.±2° C. withcontinuous, gentle agitation are generally assigned a 5 day expiry. Inour study we investigated serial (daily), collagen-induced PDGF-BBrelease from normal donor platelets stored at room temperature over 8days under variable conditions.

Collection of Donor Platelets

A total of 125.0 mL of blood was taken from a haemostaticallyasymptomatic, clinically well adult male donor into 25 BD Vacutainer®tubes (Bunzl Healthcare, Enfield, UK), each containing 1.0 mL ofAcid-Citrate-Dextrose solution B (ACD-B) as the anticoagulant.

The non-traumatic venepuncture was performed with minimal stasis using a21 g-butterfly needle. The donor had not received any drugs known toaffect platelet function in the preceding two weeks, or any foodstuffsknown to affect platelet function in the preceding five days.

Blood for platelet diagnostics is normally taken into 0.105M tri-sodiumcitrate in a ratio of nine parts blood to one part anticoagulant.However, this is based on diagnostic assays being performed within fourhours of venepuncture to assess function on freshly drawn plateletsbefore natural deterioration adversely affects diagnostic accuracy. Thechoice of ACD-B as anticoagulant for this study was based on the work ofLei et al who demonstrated that ACD-B was superior to tri-sodium citratein maintaining platelet structure integrity and preventing spontaneousaggregation.

Preparation of Platelet-Rich Plasma

The blood was allowed to cool for 15 minutes after the venepuncture toprevent formation of plasma clots. The tubes were then centrifuged at22° C. in an IEC Centra CL3 centrifuge (Thermo Scientific, Basingstoke,UK) at 130 RCF (800 RPM) for 20 minutes to generate PRP.

After centrifugation, the PRP was pooled by transferring into two 30 mLSterilin™ polypropylene universal containers (Thermo Scientific) usingplastic transfer pipettes and avoiding the white blood cell-rich buffycoat layer. Use of polypropylene containers and plastic pipettesprevents platelet activation during sample manipulation. The contents ofthe two containers were multiply inter-mixed to achieve homogeneity.

The remaining whole blood was centrifuged at 2450 RCF (3500 RPM) for 10minutes to obtain platelet poor plasma (PPP) for use as a blank in thecollagen-induced platelet activation. The PPP was transferred into aseparate universal container.

A blood count was performed on the PRP [12] using a Sysmex PocH 100i(Sysmex UK, Milton Keynes, UK) to check that:

The platelet count was between 150-600×109/L

The white blood cell count was <0.5×109/L

The red blood cell count was <0.5×1012/L

If the platelet count is too high, artefactual inhibition of in vitroaggregation can occur, and too many red blood cells will interfere withdetection of aggregation. White blood cells can inhibit plateletaggregation so it is important that most of them are removed from PRP.

Collagen-Activated Platelet Aggregometry

Immediately after preparation, aliquots of the PRP were subjected toactivation by Collagen Reagent HORM® (Takeda Austria, Linz, Austria), anequine tendon collagen suspension, in a PAP8 platelet aggregometer(Alpha Laboratories, Eastleigh, UK). The PAP8 employs lighttransmittance aggregometry, which is summarised in FIG. 5.

The platelets were activated with collagen at a final concentration inthe PRP of 5 μg/mL, a standard concentration for diagnostic purposesthat should instigate full aggregation in normal platelets. Separatealiquots were activated with collagen at a concentration of 10 μg/mL toascertain whether doubling the dose might maximise aggregation, andthus, α-granule content release. Once aggregation was complete, thealiquots were centrifuged to pellet the platelet aggregates and thesupernatant removed and frozen at −80° C.

Storage of Platelet-Rich Plasma

The remaining PRP was stored under different conditions in two types ofcontainer. Container 1 was a 70 mL Nunc™ non-treated flask (ThermoFisher Scientific, Langenselbold, Germany), a sterile, non-activatingpolystyrene tissue culture flask, which is shown in FIG. 6(a). Althoughthe polystyrene body of the flask is not oxygen permeable, the filtercap permits a constant air flow. Container 2 was a 30 mL Sterilin™polypropylene universal container (Thermo Scientific), which is sterilebut not oxygen permeable, which is shown in FIG. 6(b).

Each container type was used to store PRP in four different ways over aneight day period, as described below:

-   (a) Kept agitated throughout an 8 day storage-   (b) Gently shaken/inverted several times per day-   (c) Kept stationary throughout-   (d) Kept agitated throughout an 8 day storage+addition of PGE1

The 150 mL volume of donated blood plus anticoagulant yieldedapproximately 65 mL of PRP, which permitted storage of approximately 8.0mL of PRP in each container.

It has long been recognised that gently agitating platelets duringstorage reduces activation and debris formation. It has been shown thatan interruption of one day has negligible, measurable effect, but longerperiods can result in significant, deleterious changes. For this reason,aliquots of PRP were stored constantly agitated, intermittentlyagitated/mixed or kept stationary to assess the impact of variableagitation. An additional constantly agitated storage condition wasintroduced to include addition of prostaglandin E1 (PGE1), a naturalplatelet inhibitor that triggers an increase in cyclic adenosinemonophosphate levels which counteracts platelet activation by reducingcalcium flux. Addition of PGE1 to PRP reduces platelet activation duringstorage whilst permitting a response to most agonists during in vitroanalysis. PGE1 was added to the PRP at a final concentration of 10μg/mL, the standard concentration for diagnostic use. All PRP was storedat room temperature since cold storage activates platelets.

A Luckham R100 Rotatest Shaker (FIG. 7) was used to agitate thecontainers requiring constant agitation. The platform was rotated onsetting 5, a mid-point setting to approximately mimic that employed forplatelets being stored for transfusion. The aliquots being mixedintermittently were shaken/inverted hourly during the laboratory corehours of 09.00-17.00 h for the first five days and left undisturbed fordays six and seven as they were over a weekend.

Serial Collagen-Activated Platelet Aggregometry

At approximately the same time each day as when the freshly prepared PRPwas analysed, aliquots of mixed PRP from each of the eight containerswere separately activated in the PAP8 aggregometer with collagen at afinal concentration in PRP of 10 μg/mL and the aggregation patternsrecorded. Once aggregation was complete, the aliquots were centrifugedto pellet the platelet aggregates and the supernatants removed andfrozen at −80° C. This was undertaken on days 2-5 and day 8. Plateletcounts were also performed each day as an on-going assessment of PRPquality and platelet activation and spontaneous aggregation. Plateletcounts were performed immediately prior to aggregometry.

Analysis of Supernatants for PDGF-BB

To assess storage and release of growth factors from stored platelets,measurement of PDGF-BB was chosen as a representative marker since ithas been employed in similar studies for the same purpose, becaplerminis a recombinant version of PDGF-BB, and reagents for analysing PDGF-BBlevels are commercially available.

PDGF-BB levels in the supernatants were quantified with Human PDGF-BBPlatinum ELISA reagent kit (Affymetrix eBioscience, Hatfield, UK). Theprinciple of measurement of PDGF-BB by enzyme-linked immunosorbent assay(ELISA) is depicted in FIG. 8.

Prior to use in quantification of released PDGF-BB in the storedsupernatants, the kit was evaluated for standard curve linearity andanalytical precision. Whilst each step of the assay was performedmanually, end-point detection, standard curve generation, test raw dataand calculation of final results were all performed on a Dynex DS2™ELISA analyser (Instrumentation Laboratory, Warrington, UK).

Cell Counting of PRP on the Day of Donation

The blood cell counts on the PRP immediately after harvesting, and priorto collagen-induced platelet aggregation, are shown in Table 1.

TABLE 7 Cell counts on PRP Parameter Results Units White cell count 0.0×109/L Red cell count 0.00 ×1012/L  Platelet count 236 ×109/L

The aliquots activated with 5 μg/mL collagen achieved 93% and 85%aggregation (mean 89%). The aliquots activated with 10 μg/mL collagenachieved 91% and 84% aggregation (mean 88%). Although this revealed noincrease in the activation/aggregation responses to a doubling of thestandard collagen concentration, the 10 μg/mL collagen concentration wasadopted for the serial testing to allow for ageing of the plateletsduring the course of the experiments.

Collagen-Induced Aggregation of Stored PRP: Day 2

Collagen-induced aggregometry was performed in duplicate on aliquots ofPRP from each storage situation. The aggregation traces for PRP storedin the Nunc™ flasks are shown in FIG. 10, and the non-oxygen permeableuniversal containers in FIG. 11. Platelet count (Plt) prior toaggregometry and mean percentage final aggregation (MA) for eachduplicate are given in the legends.

Collagen-Induced Aggregation of Stored PRP: Day 3

Collagen-induced aggregometry was performed in duplicate on aliquots ofPRP from each storage situation. The aggregation traces for PRP storedin the Nunc™ flasks are shown in FIG. 12, and the non-oxygen permeableuniversal containers in FIG. 13.

Platelet count (Plt) prior to aggregometry and mean percentage finalaggregation (MA) for each duplicate are given in the legends

Collagen-Induced Aggregation of Stored PRP: Day 4

Collagen-induced aggregometry was performed in duplicate on aliquots ofPRP from each storage situation. The aggregation traces for PRP storedin the Nunc™ flasks are shown in FIG. 14, and the non-oxygen permeableuniversal containers in FIG. 15 Platelet count (Plt) prior toaggregometry and mean percentage final aggregation (MA) for eachduplicate are given in the legends.

Collagen-Induced Aggregation of Stored PRP: Day 8

Collagen-induced aggregometry was performed in duplicate on aliquots ofPRP from each storage situation. The aggregation traces for PRP storedin the Nunc™ flasks are shown in FIG. 18, and the non-oxygen permeableuniversal containers in FIG. 19 Platelet count (Plt) prior toaggregometry and mean percentage final aggregation (MA) for eachduplicate are given in the legends.

Plots of changes in % aggregation over time in variably stored PRP areshown in FIGS. 20-23.

Platelet Parameters Over Time in Variably Stored PRP

In addition to counting platelet numbers, the Sysmex PocH 100i analysergenerates other platelet diagnostic parameters, as described below:

Mean platelet volume (MPV)

Platelet distribution width (PDW) calculates the relative width ofplatelet volume distribution

Platelet-large cell ratio (P-LCR) is the ratio of platelets smaller than12 fL to those with a volume between 12-30 fL

Reference ranges from literature for whole blood are as follows:

-   MPV: 7.5-11.5 fL-   PDW: 9.3-16.0 fL-   P-LCR: 24.8-41.2%

Table 8 shows the platelet parameters in variably stored PRP immediatelyprior to collagen activation on each day.

TABLE 8 Serial platelet parameters in variably stored PRP StorageStorage Platelet Day of analysis container conditions parameters Day 1Day 2 Day 3 Day 4 Day 5 Day 6 No storage Fresh PRP PDW (fL) 11.6 — — — —— MPV (fL) 9.7 P-LCR (%) 22.3 Nunc ™ flask Constant PDW (fL) — 16.2 15.214.0 12.2 8.8 agitation MPV (fL) 11.8 11.1 10.7 10.1 8.6 P-LCR (%) 41.236.0 33.0 28.5 18.4 Intermittent PDW (fL) — 13.4 17.5 14.4 14.2 12.1agitation MPV (fL) 10.7 11.8 11.3 11.0 10.4 P-LCR (%) 29.7 41.8 36.434.7 30.5 Stationary PDW (fL) — 12.5 16.4 16.1 15.2 13.9 MPV (fL) 10.211.7 11.7 11.4 11.0 P-LCR (%) 26.4 41.5 41.2 38.0 35.6 Constant PDW (fL)— 16.5 15.2 14.1 12.2 9.7 agitation + PGE₁ MPV (fL) 11.7 11.1 10.9 10.28.9 P-LCR (%) 41.3 36.4 34.4 29.1 20.2 Universal Constant PDW (fL) — 9.99.4 No 8.6 8.3 agitation MPV (fL) 8.8 8.6 analyser 8.6 8.4 P-LCR (%)16.9 17.9 output 19.2 18.3 Intermittent PDW (fL) — 11.3 11.6 12.6 12.013.8 agitation MPV (fL) 9.6 9.7 10.2 10.1 10.7 P-LCR (%) 21.0 21.9 25.824.9 31.1 Stationary PDW (fL) — 11.0 11.8 11.5 12.3 14.4 MPV (fL) 9.510.1 10.1 10.1 11.4 P-LCR (%) 19.6 23.8 23.8 23.5 36.6 Constant PDW (fL)— 9.8 8.7 8.5 7.8 8.9 agitation + PGE₁ MPV (fL) 8.8 8.3 8.1 8.1 8.6P-LCR (%) 16.4 17.1 13.8 15.4 20.2

TABLE 9 Serial PDGF-BB levels in variably stored collagen (10 μg/ml)activated platelets Storage Storage PDGF-BB (pg/mL) container conditionsDay 1 Day 2 Day 3 Day 4 Day 5 Day 6 No storage Fresh PRP 18 734 — — — —— Nunc ™ flask Constant — 13 190 14 580 11 081 13 689 10 332 agitationIntermittent — 12 871 11 488 10 589  9 991 10 042 agitation Stationary —11 159 12 441 10 973 11 969 11 210 Constant — Not 10 055  7 842  9 796 9 808 agitation + PGE₂ available Universal Constant — Not 12 486 12 90510 950  9 925 agitation available Intermittent — 15 303 14 141 12 514 10547  8 701 agitation Stationary — 10 875 13 649 12 939 10 938 11 398Constant — 13 082 11 836  9 829 10 148  8 967 agitation + PGE₃ Note thatresults for Day 2 of constant agitation + PGE₁ in the Nunc ™ flask andconstant agitation in the universal are unavailable due to technicalproblems

Virtually without exception, publications on the use of Plasma forcosmetic and wound healing uses describe use of freshly drawn PRP, or insome cases, PRP that has been frozen and thawed. The aim of the presentstudy was to evaluate growth factor release from PRP stored at roomtemperature over a number of days, using PDGF-BB as the marker. FreezingPRP is known to damage the platelets causing a rapid release of growthfactors on thawing. For this reason frozen PRP may not be useful fortechniques where the plasma is stored for use on several consecutivedays unless the PRP is frozen in portions so that one may be thawedimmediately before each application.

Effects of Storage on Platelet Number and Size Parameters

There was a steady fall in platelet count over time in all storagecontainers and conditions except for the two universals under constantagitation which exhibited a marked fall on Day 2 before beginning asteady decline after that. There is no immediately obvious explanationfor this observation, although the platelet parameter results arerevealing. Both these containers had lower PDW, MPV and P-LCR valuesthan the stationary and intermittently agitated universals on all daysof analysis. The smaller platelet size, narrower width distribution andlower ratio suggest that larger platelets were missing from these twocontainers, but why this may be the case is unclear. It would betempting to suggest that the intensity of agitation was too high and hadachieved a degree of activation, with the more reactive larger plateletsperhaps being more susceptible, yet this was not mirrored in the twoconstantly agitated Nunc™ flasks with apparently greater oxygen access.The otherwise steady decline in platelet count in all containers willhave been largely due to the well described platelet storage lesion.

Remarkably, all PDW, MPV and P-LCR results from universal-stored PRPwere lower than their Nunc™ flask-stored counterparts, indicatingcontainer-induced effects. Furthermore, platelet parameters on Day 2were closer to those of fresh PRP in the universal-stored PRP than Nunc™flask-stored PRP. Minor increases in MPV and PDW during storage up tofive days have been reported previously but not to the marked extent ofthe PDW results in the Nunc™ flask-stored PRP. If MPV does not normallyincrease significantly over this time in stored PRP, the moderateincrease on Day 2 in these flasks accompanied by marked increase in PDW,particularly in the constantly agitated flasks, is suggestive that adegree of microaggregate formation had occurred. This would explain theincrease in P-LCR that was suggesting the presence of higher numbers oflarger cells, which were more likely to have been microaggregates. Theonly moderately increased MPVs indicate they remained small enough forthe vast majority to be counted as platelets. Interestingly, PDW, MPVand P-LCR reduced over time in the constantly agitated Nunc™ flasks,probably due to increasing spontaneous aggregation over time such thatthe largest aggregates were not aspirated into the blood count analyser.Further evidence of microaggregate formation was found from apparent lowred blood cell counts in many of the Nunc™ flask-stored PRP results(data not shown). There were no red cells in the PRP on Day 1 and thesecounts were due to small populations of aggregates too large to becounted as platelets. The only universal-stored PRP to register a lowred cell count was from the stationary universal on Day 8, which had thehighest PDW, MPV and P-LCR values of any universal-stored PRP. MPV wasmore stable over time in the intermittently agitated and stationaryNunc™ flasks, suggesting that constant agitation exacerbated spontaneousaggregate formation in a storage situation where low-level activationwas already more likely. There was little difference between theplatelet parameters of the constantly agitated Nunc™ flasks with andwithout PGE1, or the universals, so it appears that the PGE1 did notsuppress spontaneous aggregation. It may be that a higher concentrationwas required, or that the PGE1 is insufficiently stable for stored PRP.

Converse results were seen in universal-stored PRP in that PDW, MPV andP-LCR were lower in the constantly agitated universals than in theintermittently agitated and stationary universals. As described above,there was a curious fall in platelet count, PDW, MPV and P-LCRparameters on Day 2 in the constantly agitated universals, which mayhave been due to an immediate, more intense formation of aggregates thatwere not counted in the analyser, followed by relatively smallreductions in PDW and MPV over time. In contrast to the steep falls inP-LCR over time in the constantly agitated Nunc™ flasks, the counterpartuniversals exhibited mild increase over time, suggesting low levelaggregate formation. Some degree of activation and aggregate formationis expected in stored platelets. Only the intermittently agitated andstationary universals mirrored baseline PDW, MPV and P-LCR values on Day2, with the anticipated gradual but small increases over time of PDW andMPV. The increases in P-LCR over time were more marked, suggesting adegree of aggregate formation in these containers too.

Based on these observations, the intermittently agitated universalmaintained the greater platelet integrity over time. Hunter et al showedthat interruption of agitation for one day produces no measurableplatelet damage, so the intermittent agitation during laboratory corehours and less than 24 hour interruption overnight appears to have beensufficient to maintain comparable integrity.

Effects of Storage on Platelet Aggregation Responses

As previously reported, there was an overall continuous fall in plateletaggregability over time under all storage conditions in the study. Anunanticipated finding was the remarkable fall in final percentageaggregation of Nunc™ flask-stored PRP on Day 2 and thereafter, in starkcontrast to the more gradual declines in universal-stored PRP. Theminimal platelet count reductions, other than from the constantlyagitated universals on Day 2, indicate that relatively few platelets hadformed aggregates and that the aggregation responses were a result ofactivation of the majority of the stored platelets. In which case, thestorage conditions in the Nunc™ flasks were detrimental to overallplatelet function.

Polypropylene is the preferred material for platelet storage althoughplatelet adhesion to untreated polystyrene is normally minimal withnon-activated platelets. However, binding of plasma proteins such asfibrinogen and VWF to the polystyrene can promote platelet adhesion andsubsequent activation. Again, the minimal fall in platelet countssuggest this either did not occur, or occurred only minimally. Thus, thecontainer itself would not be expected to inhibit platelet function andconditions in the PRP itself were the more likely culprit. The mostprobable explanation for such marked storage-induced differences ininitially identical samples of PRP is a drift in plasma pH. Watts et alshowed that PRP stored in a closed system to maintain pH betterpreserved platelet function than PRP stored in a controlled CO2/airenvironment, which was probably related to the presence of an air/liquidinterface in the latter. The universals have screw top lids which wereonly removed once a day to extract PRP for aggregometry, whilst theNunc™ flasks permitted air flow. Since the 30 mL universals were used tostore only approximately 8.0 mL of PRP, they likely retained sufficientoxygen in the ‘dead space’ until the next opening to not significantlyimpair platelet integrity. The anticoagulant used for sample collectioncan also affect pH of PRP.

A commonality in aggregometry responses between the two storagecontainers is the reduced final percentage aggregation in PRP from theconstantly agitated samples compared to those intermittently agitated orkept stationary. This is counter-intuitive as accepted dogma is thatstored platelets should be constantly, gently agitated to reduceactivation and debris formation. It may be that the agitation was tooharsh or too gentle for the PRP volumes and containers that wereemployed, or that pH changes were exaggerated by the constant movement,even in the universals since they retained an appreciable amount of air.

Effects of Storage on PDGF-BB Levels

The amount of released PDGF-BB in freshly prepared PRP is affected bynumerous variables, including anticoagulant, donor whole blood plateletcount, PRP preparation conditions, platelet count in PRP and type andconcentration of agonist used to induce release. Nonetheless, thePDGF-BB level of 18 734 pg/mL in the freshly prepared PRP in this studybroadly mapped to levels reported by other workers. From similarcentrifugation conditions, Gonzalez et al reported a PDGF-BB range of9.6-14.6 ng/mL from PRP obtained from 32 donors, and Castillo et alreported 13.0-33.2 ng/mL from five donors.

A marked fall in PDGF-BB levels was observed on Day 2 in all storageconditions where results were available. The mean percentage reductionwas 32%, ranging from the 18% reduction in the intermittently agitateduniversal to the 42% reduction in the stationary universal. Most werefollowed by a steady, continuous decrease in PDGF-BB levels, someostensibly reaching a plateau by Days 5 and 8. To a large extent thesefindings are unsurprising as platelet secretory capacity falls duringstorage, yet by Day 8, appreciable levels of PDGF-BB remained despitethe expectation that the majority of platelets would be dead or poorlyfunctional by that time. A potential explanation is that the PDGF-BB inthe supernatant may have merely leaked from the platelets as they agedand membrane integrity was lost. In view of the minimal reduction inplatelet counts for most storage conditions, and even the steady fallafter the initial marked reduction in the constantly agitateduniversals, this would seem unlikely to be the only causative factor.Furthermore, if the platelets were fragmenting over time there wouldlikely have been gradual reductions in MPV, which was only observed inconstantly agitated Nunc™ flasks, whilst others remained stable orincreased over time. However, microaggregate formation could mask moresubtle volume loss from membrane fragmentation. If those MPV reductionsin the constantly agitated Nunc™ flasks were due to fragmentation, aconcomitant increase in PDW over time would be expected, but theconverse was the case.

What is also interesting is that the relatively poor aggregationresponses from Day 2 onwards in constantly agitated Nunc™ flask-storedPRP are not accompanied by similarly marked PDGF-BB reductions. This wastrue, albeit to a lesser extent, with the other paired storageconditions. It appears that some of the PDGF-BB in the supernatants isnot derived from platelet release, and the explanation for this comesfrom other studies that additionally measured PDGF-BB in PPP. Gonzalezet al reported a range in PPP of 8.8-12.6 ng/mL, whilst Valeri et alreported a range of 1549-2813 pg/mL. There are clear between-studydifferences yet plasma PDGF-BB does contribute to values measured inplatelet releasates unless platelets are washed prior to analysis. Themechanical trauma of PPP and PRP preparation will also have contributedto the presence of free PDGF-BB in the PPP. The platelet counts in thePRP of the study by Gonzalez et al were adjusted to 250×109/L, similarto the count in the donor PRP for this study, whilst the mean count inPRPs from the study by Castillo et al was 566×109/L. From a clinicalperspective, the PDGF-BB values from collagen-activated PRP reported inthis study relate to a lower platelet count than would be used inclinical practice, so the differentiation between release from fresh andstored PRP would be greater. Again, the aim of the study was toinvestigate relative loss of PDGF-BB release, which had to be performedat lower platelet counts to maximise application of available analyticaltechniques.

Parameter Stability

Platelet rich plasma stored in the intermittently agitated universalmaintained better stability in the parameters studied than that storedin other conditions. Up to Day 5 there was minimal reduction in plateletcount and elevations in PDW, MPV and P-LCR, particularly in comparisonwith some of the marked platelet parameter changes in PRP stored inNunc™ flasks. The platelet changes in the stationary universal weregreater except the platelet count, and there is no clear explanation forthe marked fall in platelet count on Day 2 in the two constantlyagitated universals.

Platelet aggregation responses to collagen activation were lower inNunc™ flask-stored PRP than universal stored PRP for the reasonsdescribed above. Higher final percentage aggregation over time wasachieved by the PRP in intermittently agitated and stationary universalscompared to their constantly agitated partners, other than an apparentsurge on Days 5 and 8 in the universal without PGE1. However, the fallsin aggregation (representing overall platelet reactivity), particularlyin Nunc™ flask-stored PRP, were not accompanied by similarly dramaticreductions in supernatant PDGF-BB levels. It seems likely that althoughsecretory capacity reduced over time, PDGF-BB levels did not fall tonear zero due to a mixture of leakage from senescing platelets,mechanical trauma and innate plasma levels.

Although PRP stored in the intermittently agitated universal maintainedbetter overall stability, it did in fact generate the lowest PDGF-BBvalue on Day 8 despite achieving 39% final aggregation. An importantconsideration is that the study merely assessed PDGF-BB concentrationbut not function, so it is impossible to know what levels of PDGF-BB aretherapeutically efficacious, and therefore, at what point during storagethe PRP should be discarded. Furthermore, the PDGF-BB itself may losefunctionality over time and be therapeutically ineffective despite anapparently high level of release.

Consideration of function aside, PDGF-BB levels from activation of PRPunder all storage conditions were within the range reported by Gonzálezet al for fresh PRP with a similar platelet count, up to Day 5. Althoughthis may be encouraging in terms of storage potential, all the valueswere markedly lower than for fresh PRP. Without knowledge of PDGF-BBfunctionality, or that of other released growth factors, it would seemthat freezing either the fresh PRP or the post-activation supernatantmight generate a more efficacious therapeutic product. Whilstacknowledging the controversy regarding PRP cryopreservation, Roffi etal showed that frozen-thawed PRP without prior agonist activationachieved comparable in vitro effects on cultured cells to those of freshPRP despite reductions in PDGF-AB/BB levels. The freeze/thaw cycle actsas a surrogate ‘activator’ by disrupting platelet membranes and inducingcontent release. In view of the lower growth factor levels in thefreeze/thawed PRP, it may be a less efficient approach to increasinggrowth factor availability than agonist-induced platelet activationsince they are ‘packaged’ within α-granules rather than free in thecytoplasm. Durante et al reported PDGF-BB levels in fresh PRP of 17904±1346 pg/mL but reduced levels even after three freeze/thaw cycles.This suggests that freezing the post-collagen activation supernatant ismore likely to generate and preserve higher growth factor levels, withthe possible additional value of excluding platelet debris from thefinal product.

The invention claimed is:
 1. A method of improving the cosmeticappearance of skin of a subject, wherein the method comprises: a)providing a sample of platelet rich plasma from the subject, wherein theplatelet rich plasma comprises more than 500,000 platelets/μl; b)providing an activator; c) admixing a portion of the platelet richplasma and a portion of the activator to form a topical composition; d)applying the topical composition to an area of the subject's skin lessthan 4 hours after the platelet rich plasma and the activator have beenadmixed to form the topical composition; and e) repeating steps (c) and(d) at least once a day for at least two days.
 2. The method of claim 1,wherein the topical composition is applied to an area of the subject'sskin less than 1 hour after the platelet rich plasma and the activatorhave been admixed to form the topical composition.
 3. The method ofclaim 2, wherein the topical composition is applied to an area of thesubject's skin less than 10 minutes after the platelet rich plasma andthe activator have been admixed to form the topical composition.
 4. Themethod of claim 3, wherein the topical composition is applied to an areaof the subject's skin less than 2 minutes after the platelet rich plasmaand the activator have been admixed to form the topical composition. 5.The method of claim 1, wherein steps (c) and (d) are repeated for atleast three days.
 6. The method of claim 5, wherein steps (c) and (d)are repeated at least once a day for at least eight days.
 7. The methodof claim 1, wherein the area of the subject's skin has been treatedusing PRP injection or another cosmetic treatment.
 8. The method ofclaim 1, wherein the platelet rich plasma comprises more than 900,000platelets/μl.
 9. The method of claim 1, wherein each time a portion ofthe platelet rich plasma is admixed with a portion of the activator toform a topical composition growth factors are released from platelets inthe platelet rich plasma so that fresh growth factors are applied to theskin each time the topical composition is applied to the skin.
 10. Themethod of claim 1, wherein the activator does not comprise calciumchloride.
 11. The method according to claim 1, wherein the activator iscollagen.
 12. The method according to claim 1, wherein the platelet richplasma is stored for at least 1 day before it is admixed with theactivator to form a topical composition.
 13. The method according toclaim 1, wherein the activator is disposed in a cosmetically acceptablecarrier.
 14. The method according to claim 13, wherein the cosmeticallyacceptable carrier comprises a cream, gel, serum, balm, sun cream, aftersun cream, foundation, tinted cream, tinted sun cream, soothing antiredness cream with green tint, eyelash gel, lip balm, scalp serum,solution, suspension, emulsion, ointment, foam, paste, lotion, powder,soap, surfactant-containing cleansing oil or spray, or a combinationthereof.
 15. The method according to claim 1, wherein the sample ofplatelet rich plasma does not have an activator added to it prior tostep (c).